Chapter 8 Detection of Fusion Events in Mammalian Skeletal Muscle Frank Suhr Abstract Cell fusion events are essential for the maintenance of skeletal muscle tissue and during its repair processes after damage. However, these mechanisms have not come much into focus in the recent years. Different methods can be used to assess ongoing cell fusion events in adult skeletal muscle tissue. Among these methods, confocal microscopy, western blotting, and quantitative polymerase chain reactions are ideal, since they provide concerted information about cell fusion events going on in skeletal muscle tissue at both qualitative and quantitative levels. Confocal microscopy allows for the visualization of exact localizations of cell fusion events in adult skeletal muscle. Western blotting allows for a semiquantitative evaluation of protein levels involved and associated with cell fusions events. Finally, quantitative polymerase chain reaction is a valuable tool to precisely assess mRNA levels of genes involved and associated with cell fusions events. In addition to the investigation if cell fusion markers in skeletal muscle tissue, in vitro cell culture systems (e.g., C2C12 cells) can be used to study cell fusions events in a highly standardized system in order to obtain detailed information about genes and proteins involved in these processes. Here, confocal microscopy, western blotting, and quantitative polymerase chain reaction are described as methods to investigate cell fusion events and how a C2C12 cell culture system can be run to support the studies of adult muscle tissue. Key words Syncytin, Myogenic progenitor cell, Confocal microscopy, Western blot, qPCR
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Introduction Mammalian skeletal muscle tissue owns a remarkable ability to regenerate upon damage and thus shows a highly plastic morphology [1]. The regenerative capacity of mammalian skeletal muscle tissue is predominantly driven by a myogenic progenitor cell population known as satellite cells [2]. Myogenic progenitors are interposed between the basal lamina and the sarcolemma of adult muscle fibers [3]. Upon tissue damage, e.g., after exhaustive mechanical exercise or during muscle disease, these myogenic progenitors are able to become activated and to enter the cell proliferation cycle, to divide (asymmetrically), to differentiate, and to fuse with adjacent muscle fibers [4]. Therefore, intercellular
Kurt Pfannkuche (ed.), Cell Fusion: Overviews and Methods, Methods in Molecular Biology, vol. 1313, DOI 10.1007/978-1-4939-2703-6_8, © Springer Science+Business Media New York 2015
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fusions among mononucleated myogenic progenitors with each other and among myogenic progenitors with multinucleated adult muscle fibers are required for the development and specifically for the maintenance of intact and integral adult muscle fibers [5, 6]. The formation of syncytia thus is an integral process to maintain functional muscle fibers in the adult mammalian organism. Different methodological approaches can be used to study fusion events in mammalian skeletal muscle tissue. Microscopic approaches, such as confocal microscopy, have the advantage to visualize ongoing fusion events in cell cultures or in adult skeletal muscle tissues allowing for the identification of the exact localization of the event, while a quantification of ongoing fusion events is impossible. Cell fusion events are complex and concerted events making a systemic evaluation in adult skeletal muscle tissue difficult, but not impossible. In order to produce as clear ideas as possible about cell fusion events, a combination of in vitro cell culture systems and ex vivo skeletal muscle tissue analyses should be preferred. Classical protein and mRNA analysis approaches, such as western blot and quantitative real-time polymerase chain reaction (qPCR), are perfect tools to quantify cellular fusion events in cell cultures, e.g., C2C12 cells. Amongst in vitro systems, adult skeletal muscle tissues isolated from rodents or humans can be harvested, e.g., after in vivo mechanical stimulation experiments inducing microdamage in skeletal muscles. Due to these impacts, skeletal muscle tissues are susceptible for the induction of cell fusion events, why microscopic, western blot and qPCR analyses are promising tools to evaluate the levels of markers reflecting cell fusion events present in skeletal muscle tissue. However, using these approaches, it is not possible to exactly localize the fusion events, why high-resolution confocal microscopy provides the power to precisely localize the spots of cell fusion events. Fusion events in mammalian skeletal muscle tissue have not been a broad focus in the literature, yet, why the knowledge about classical fusion markers is still limited; however, there are some proteins known to be involved in fusion events in skeletal muscle tissue [6, 7]. Among these proteins, the family of syncytin proteins, mostly known as critical players in placentogenesis [8], seems to be involved in cellular fusion initiated in adult skeletal muscle tissue. These markers and also related ones can be assessed by microscopic and western blot/qPCR methods in order to investigate cell fusion events in adult skeletal muscle tissue. Herein, I will describe methodological approaches to study fusion events in mammalian skeletal muscle tissue. I will describe syncytin-1 analysis in both cell culture of myogenic progenitors and skeletal muscle tissue from mice stressed in vivo by mechanical stimulation.
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Materials Prepare all solutions using ultrapure water and analytical grade reagents. Use only sterile plastic consumables. Autoclave solutions etc. before use.
2.1
Cell Culture
1. C2C12 cells. 2. Uncoated cell culture flasks. 3. C2C12 proliferation medium (500 mL): 370 mL DMEM, 100 mL 20 % fetal calf serum (FCS), 5 mL 1 % penicillin/ streptomycin (100× stock solution, Invitrogen Life Technologies), 10 mL 4 mM L-glutamine, 10 mL 1.5 g/L sodium bicarbonate, 5 mL 1 mM sodium pyruvate. 4. C2C12 differentiation medium (500 mL): 450 mL DMEM, 20 mL 4 % horse serum, 5 mL 1 % penicillin/streptomycin, 10 mL 4 mM L-glutamine, 10 mL 1.5 g/L sodium bicarbonate, 5 mL 1 mM sodium pyruvate. 5. Sterile cell culture bench. 6. Cell culture.
2.2 Confocal Microscopy
1. Tris-buffered saline (TBS): 0.05 M Tris-base, 0.15 M sodium chloride (NaCl), use 1 N hydrochloric acid to adjust pH to 7.6. 2. Phosphate-buffered saline (0.2 M PBS): 28.8 g water-free disodium hydrogen phosphate, 5.2 g sodium hydrogen phosphate-monohydrate, 17.5 g sodium chloride, pH 7.4. 3. 4 % PFA fixative: dissolve 4 g paraformaldehyde in 40 mL ultrapure water under heating (60 °C, avoid cooking), clear the solution by dropwise addition of 1 N hydrochloric acid, filtrate the solution afterwards, mix with 50 mL 0.2 M PBS, adjust pH to 7.4, add ultrapure water to 100 mL. 4. Cryo microtome (Leica CM 1900). 5. Microscope slides, poly-Lysine coated. 6. 3 % hydrogen peroxide solution in methanol: Mix 0.075 mL 30 % hydrogen peroxide solution with 0.675 mL ultrapure water and 3 mL methanol. 7. 0.5 M ammonium chloride-TBST solution: Dissolve 0.59 g ammonium chloride in 20 mL TBS and add 50 μL Triton-X 100. 8. 5 % bovine serum albumin solution (15 mL): Dissolve 0.75 g bovine serum albumin (BSA) in 15 mL ultrapure water. 9. 0.8 % bovine serum albumin solution (15 mL): Dissolve 0.12 g bovine serum albumin (BSA) in 15 mL ultrapure water. 10. Primary polyclonal syncytin-1 antibody produced in rabbit (Abnova).
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11. Secondary antibodies Alexa Fluor® 555 goat anti-rabbit (Invitrogen Life Technologies). 12. DNA staining dyes: 4′,6-Diamidino-2-Phenylindole dihydrochloride (DAPI) nuclear acid staining dye, NucRed® Live 647 Ready probes reagent (Draq5) nuclear acid staining dye (Invitrogen Life Technologies). 13. Aqua-Poly/Mount water-soluble, non-fluorescing mounting medium (Polysciences). 14. Confocal microscope (e.g., LSM 510Meta equipped with one NeHe laser generating a laser line with a wavelength 543 nm and one NeHe laser generating a laser line with a wavelength 633 nm). 2.3 Protein Isolation from Skeletal Muscle Tissue
1. 20–30 mg skeletal muscle tissue. 2. Liquid nitrogen (N2). 3. Mortar and pestle. 4. Cell lysis buffer (Cell Signaling). Add PMSF (1:100 v:v) and phosphatase inhibitor (1:50 v:v) to commercially available 1× cell lysis buffer. 5. 100 μM PMSF and 50 μm phosphatase inhibitor (Thermo Scientific). 6. SDS polyacrylamide gel electrophoresis: Sterile 1.5 mL Eppendorf tubes.
2.4 Protein Content Determination
1. Bradford protein assay kit.
2.5 SDS Polyacrylamide Gel Electrophoresis
1. SDS-PAGE running buffer: 0.025 M Tris-base, 0.192 M glycine, 10 % (m:v) sodium dodecylsulfate (SDS), pH 8.3. 2. 30 % acrylamide/Bis-acrylamide solution (37.5:1 acrylamide– bis). Commercially available ready-to-use solution. 3. 10 % sodium dodecylsulfate (SDS) solution: 10 g SDS dissolved in 100 mL ultrapure water, agitate very carefully to prevent excess foaming. 4. 10 % ammonium peroxodisulfate (APS) solution: 10 g dissolved in 100 mL ultrapure water. Store at 4 °C for no longer than 4 weeks. 5. Resolution gel buffer: 1.5 M Tris-base, pH 8.8. Store at room temperature. 6. Stacking gel buffer: 0.5 M Tris-base, pH 6.8. Store at room temperature. 7. Sample lysis buffer (2× Laemmli buffer): 0.125 M Tris-base, 4 % SDS, 10 % β-mercaptoethanol, 20 % glycerol, 0.004 % bromophenol blue, pH 6.8. Store aliquots frozen at −20 °C. 8. N,N,N′,N′-tetramethylethylenediamine (TEMED), commercial ready-to-use solution.
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Western Blot
1. Polyvinylidene fluoride (PVDF) membrane. 2. Tris-buffered saline (TBS, 10×, 2 L): 121.40 g Tris base, 175.32 g NaCl, adjust pH to 7.6 using HCl. 3. TBST: TBS containing 0.05 % Tween-20. 4. Blocking solution: 5 % milk powder in TBST. Prepare freshly immediately before use. 5. Primary and secondary antibody diluents solution: 5 % bovine serum albumin in TBST. 6. Mini PROTEAN® Bio-Rad).
tetra-cell
system
(1.5
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7. Trans-Blot Turbo system (Bio-Rad). 8. Western blot transfer buffer (Towbin buffer): 0.025 M Trisbase, 0.192 M glycine, pH 8.3. Immediately before use add 5–20 % (v:v) methanol. 9. Protein standard ladder. 10. Whatman blotting paper. 11. Primary polyclonal syncytin-1 antibody produced in rabbit (Abnova). 12. Primary monoclonal glyceraldehyde-3-phosphate dehydrogenase (Gapdh) antibody raised in rabbit (Cell Signaling). Gapdh can be used as a “housekeeping” protein. 13. Secondary antibody: Stabilized goat anti-rabbit IgG (H + L), peroxidase-conjugated (Thermo Scientific). 14. Enhanced chemiluminescence (ECL) western blotting substrate kit. 2.7 mRNA Isolation from Skeletal Muscle
1. 20–30 mg skeletal muscle tissue. 2. Liquid nitrogen (N2). 3. Mortar and pestle. 4. Sterile 2.0 mL tubes. 5. TriReagent 6. 1-bromo-3-chloropropane. 7. Ethanol, molecular grade. 8. 3 M Sodium acetate, adjust pH to 5.5 by glacial acetic acid. 9. RNase-free water. 10. Agarose. 11. HDGreen Plus DNA stain to visualize RNA (Intas).
2.8 Quantitative Polymerase Chain Reaction (qPCR)
1. Omniscript RT Kit for cDNA synthesis (Qiagen). 2. Barrier Tips 1,000, 200, 100, 10 μL (low binding tips). 3. DNA low binding tubes 0.5 and 1.5 mL.
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4. PCR 96 tube plates. 5. Strips of eight PCR caps. 6. QuantiTect SYBR Green PCR Kit (Qiagen). 7. Syncytin-1 primers (forward: CAT CTA TGC TGG ATG AAG CCT, reverse: AGA CCC TGG CAT GGC CAT TA) [8]. 8. Gapdh primers (forward: GAC ATG CCG CCT GGA GAA AC, reverse: AGC CCA GGA TGC CCT TTA GT) [9]; Gapdh can be used as a “housekeeping” gene. 9. Thermocycler. 10. 10× MOPS (3-(N-morpholino)propanesulfonic acid): dissolve 41.8 g of MOPS in 700 mL ultrapure water, adjust pH to 7.0 with 2 N NaOH, add 20 mL 1 M sodium acetate and 20 mL of 0.5 M EDTA, adjust the volume to 1 L buffer and leave the flask in the water bath, mix the solution; to make 1× MOPS buffer dilute (100 mL 10× MOPS buffer in 900 mL ultrapure water). 11. Northern Max® formaldehyde loading dye (Life Technologies).
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Methods In the following, different methods are described to establish and run a C2C12 cell culture system in the lab for supportive experimental approaches for the study of cell fusion events in adult skeletal muscle tissue, to perform high quality confocal microscopy, to perform western blotting, and to perform quantitative polymerase chain reaction.
3.1
Cell Culture
1. Keep C2C12 cells in cell culture flasks at 37 °C and 5 % CO2 in proliferation medium. 2. For experimental procedures plate C2C12 cells on gelatinecoated (0.1 % in PBS) glass cover slips or petri dishes at a density of 10,000 cells/cm2. 3. Keep C2C12 cells in proliferation medium until 80–90 % confluence is reached. 4. Harvest proliferating C2C12 cells for further experiments (e.g., confocal microscopy, western blotting, quantitative polymerase chain reaction). 5. Switch medium to differentiation medium and maintain C2C12 up to 10 days in culture. 6. Change media every second day [10].
3.2 Confocal Microscopy
1. Store the skeletal muscle tissue to be analyzed for at least 20 min at −20 °C within the cryo microtome. 2. Cut 7 μm slices of the skeletal muscle tissue by means of the cryo microtome and mount the slices on the microscope slide.
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Place at least three consecutive slices on one microscope slide to enable the staining of positive slices and also negative controls. 3. Use a grease pencil to edge each single slice mounted on the microscope slide. This can be repeated several times when the edge is removed during incubation steps. 4. Wash the slices three times 5 min with TBS. 5. Block endogenous peroxidases. Incubate the slices with 3 % hydrogen peroxide solution for 10 min. 6. Wash the slices three times 5 min with TBS. 7. Permeabilize the skeletal muscle sarcolemma and unmask the epitopes by incubation of the slices with Triton-X 100 solution. 8. Wash the slices three times 5 min with TBS. 9. Blocking of unspecific binding epitopes with 5 % bovine serum albumin solution. 10. Incubate the slices with the primary antibody against syncytin1 (dilution 1:100) dissolved in 0.8 % bovine serum albumin solution. Incubate for at least 16 h overnight at 4 °C. 11. Use one slice on each microscope slide as negative control. Incubate this slice with 0.8 % bovine serum albumin without the primary antibody against syncytin-1 (End of day 1). 12. Wash the slices three times 5 min with TBS. 13. Incubate all slices with the secondary antibody Alexa Fluor® 555 goat anti-rabbit (dilution 1:500) dissolved in TBS solution for at least 60 min at room temperature. 14. Wash the slices three times 5 min with TBS. 15. Incubate all slices with DNA staining dyes: 4′,6-Diamidino-2Phenylindole, dihydrochloride (Dapi) nuclear acid staining dye (dilution 1:10,000) and NucRed® Live 647 Ready probes reagent (Draq5) nuclear acid staining dye (dilution in 1:10,000) dissolved in TBS solution. 16. Mount microscope slides with Aqua-Poly/Mount mounting medium. 17. Evaluate the slices by means of a confocal microscope (Fig. 1). 3.3 SDS Polyacrylamide Gel Electrophoresis/ Western Blot 3.3.1 Tissue Homogenization and Sample Preparation
1. Prepare 1.5 mL tubes containing 200 μL 1× cell lysis buffer; name the tube. 2. Cool the tubes on crushed ice. 3. Use mortar and pestle to homogenize skeletal muscle tissue to a fine powder in liquid nitrogen (N2). 4. Transfer skeletal muscle powder carefully to the prepared 1× cell lysis buffer-containing tube.
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Fig. 1 Syncytin-1 immunofluorescence staining in skeletal muscle tissue. (a) Syncytin-1 (stained by means of Alexa 555) is expressed at low levels at sedentary conditions. (b) After 6 weeks to chronic exercise, syncytin-1 is pronouncedly increased in skeletal muscle tissue (Tissue from Suhr et al., submitted). Bar = 100 μm
5. Vortex at least for 20 s to dissolve the powdered skeletal muscle tissue slowly in lysis buffer. 6. Spin shortly to remove cell lysis buffer from the lid of the tubes. 7. Vortex roughly for 30 s. 8. Spin at 9,000 × g for 5 min at 4 °C. 9. Carefully transfer the supernatant into a freshly prepared 1.5 mL tube. 10. Spin supernatant at 9,000 × g for 20 min at 4 °C. 11. Carefully transfer the supernatant into a freshly prepared 1.5 mL tube. 12. Measure protein concentration by means of the Bio-Rad Bradford protein assay kit. 13. Dilute samples with 2× Laemmli buffer to the respective needed protein concentration. Afterwards, heat the lysate/ Laemmli buffer mixture up to 95 °C for 5 min. 14. Let the samples cool down to room temperature and centrifuge the samples to remove condensates from the lid and the edge of the tubes. 15. Store samples on ice when they are processed immediately or store at −80 °C until usage.
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1. Handle the Mini PROTEAN® tetra-cell system as instructed by the manual, use 1.5 mm spacer. 2. Pour a 10 % acrylamide-containing gel (10 mL solution): 3.3 mL ultrapure water, 2.7 mL 30 % acrylamide solution, 2.5 mL resolution gel buffer, 100 μL 10 % SDS, 10 % APS, 5 μL TEMED. 3. Cover the gel with 500 μL isopropanol. 4. Let the gel polymerize for at least 30 min at room temperature. 5. In between pour a 5 % stacking gel (5 mL solution): 3.40 mL ultrapure water, 0.83 mL 30 % acrylamide solution, 0.63 mL resolution gel buffer, 50 μL 10 % SDS, 50 μL 10 % APS, 5 μL TEMED. 6. Remove the isopropanol from the resolution gel and pour the stacking gel on top of the resolution gel. 7. Place the comb immediately on top of the freshly poured stacking gel. 8. Let the gel polymerize for at least 30 min at room temperature. 9. Place the gel-containing tray into the tank and fill the gel electrophoresis buffer into both the tank and the gel-containing tray. 10. Carefully remove the comb from the stacking gel and carefully rinse the slots with gel electrophoresis buffer using a Pasteur pipette. 11. Carefully load protein marker standards into the respective slot. 12. Carefully load the samples into the respective slot. Usually, 15–20 mg of total protein is sufficient for gel electrophoresis coupled to western blot procedures (see Note 1). 13. Start running the gel at constant 80 V to allow samples to accumulate in the stacking and to transmigrate into the resolution gel (takes around 15 min). Afterwards increase the electrical potential to 140 V in order to transmigrate the samples through the resolution gel. 14. Stop running the gel when the resolution of the gel fits with the experimental setup (takes around 45–60 min), e.g., optimal resolution range for the detection of syncytin-1 or other proteins of interest. 15. Take out the gel-containing tray from the tank and process the gels further for western blot (see below).
3.3.3 Western Blot
1. Soak Whatman papers in freshly prepared 1× transfer buffer (Towbin buffer).
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2. Activate PVDF membrane in 100 % methanol for 10 s, afterwards deposit PVDF membrane in freshly prepared 1× transfer buffer (Towbin buffer). 3. Prepare the semidry blotting apparatus (e.g., Trans-Blot Turbo system, Bio-Rad) as indicated in the manual. Arrange Whatman papers, PVDF membrane and polyacrylamide gel as follows (from bottom, cathode, to top, anode): three Whatman papers, PVDF membrane, polyacrylamide gel, two Whatman papers. When stacking the Whatman papers, PVDF membrane and polyacrylamide gel on top of each other as indicated, make sure that no air bubbles remain between individual layers. Otherwise a constant current field cannot pass the layers resulting in lower signal detection. 4. Run the semidry blotting apparatus (e.g., Trans-Blot Turbo system, Bio-Rad) as indicated in the manual for middle-large proteins (see Note 2). 5. After finishing the run of the semidry blotting apparatus (e.g., Trans-Blot Turbo system, Bio-Rad) cut PVDF membrane in two parts: one part to enable for example syncytin-1 detection (around 55 kDa) and one part to enable Gapdh detection (around 36 kDa). 6. Incubate PVDF membranes in 5 % milk powder blocking solution for at least 90 min at room temperature. The standard protein ladder should be visible on PVDF membranes (see Note 3). 7. Prepare primary antibody solutions: dissolve the primary antibody against syncytin-1 raised in rabbit in 5 % bovine serum albumin solution (dilution 1:200); dissolve the primary antibody against Gapdh raised in rabbit in 5 % bovine serum albumin solution (dilution 1:15,000). 8. Incubate/hybridize PVDF membranes for at least 16 h overnight at 4 °C with the respective diluted primary antibody solution. 9. Wash with TBST solution at least three times 10 min at room temperature. 10. Incubate/hybridize PVDF membranes with secondary antibody solutions: dissolve secondary antibody stabilized goat anti-rabbit IgG (H + L), peroxidase-conjugated (dilution 1:2,000) in TBST and incubate PVDF membranes for at least 60 min at room temperature. 11. Wash with TBST solution at least three times 10 min at room temperature. 12. Incubate/hybridize PVDF membranes with enhanced chemiluminescence (ECL) western blotting substrate kit for 2–3 min. 13. Visualize PVDF membranes using either an imaging system (e.g., Chemidoc, Bio-Rad) or X-ray films in a dark room (Fig. 2).
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Fig. 2 Syncytin-1 expression measured by western blot in skeletal muscle tissue. Syncytin-1 is expressed at low levels at sedentary (Sed) conditions. After 6 weeks to chronic exercise (Exerc), syncytin-1 is pronouncedly increased in skeletal muscle tissue. These data correspond to the data shown in Fig. 1. Gapdh was used as a “housekeeping” protein (Tissue from Suhr et al., submitted)
3.4 Quantitative Polymerase Chain Reaction
1. Prepare 2 mL tubes containing 1,000 μL TriReagent, name the tube.
3.4.1 RNA Isolation: Tissue Homogenization
3. Use mortar and pestle to homogenize skeletal muscle tissue to a fine powder in liquid nitrogen (N2).
2. Cool the tubes on crushed ice.
4. Transfer skeletal muscle powder carefully to the prepared TriReagent-containing tube. 3.4.2 RNA Isolation: Purification
1. Spin shortly to remove TriReagent from the lid of the tubes. 2. Add the volume (BCP) to the TriReagent solution in a 1:10 ratio. 3. Vortex tubes for 15 s. 4. Leave at RT for 2–15 min. 5. Spin at 12,000 × g for 15 min at 4 °C. The solution separates: upper aqueous phase (RNA), interphase (DNA), lower red phase (proteins and RNases). 6. Transfer most of the aqueous phase (450 μL) to a fresh 1.5 mL tube. Avoid contact with interphase and lower phase. In case of contact, spin again. Interphase and organic phase may be stored at −80 °C for further purifications. 7. Add an exactly equal volume of isopropanol to the aqueous phase to precipitate the RNA. Mix by inversion. 8. Leave at RT for 5–10 min. 9. Spin at 12,000 × g for 8 min at 4 °C. Place the tube upwards. The pellet should be visible after spinning.
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10. Remove the supernatant (about 900 μL). 11. Add an equal volume of 75 % EtOH (RNase free) per volume (TriReagent). Wash sides and lid by inversion. 12. Spin at 7,500 × g for 5 min at 4 °C. 13. Remove the supernatant. 14. Spin at 7,500 × g for 1 min at 4 °C. 15. Remove the remaining supernatant, about 10 μL (see Notes 4 and 5). 16. Air-dry the pellet for 5 min, but do not dry the pellet completely as it will be difficult to get resuspended. 17. Dissolve the pellet in RNase-free water. Vortex and spin shortly at the end. 18. Measure RNA concentration and purity by means of for example NanoDrop machine (Thermo Scientific) following the manuals for RNA measurement. 19. RNA concentration should yield about 200–1,000 ng/mL. 20. 260/280 ratio should be 1.8–2.0, 260/230 ratio should be 2.0–2.2 in order to guarantee high RNA integrity and purity (for details see manuals of the NanoDrop machine). 3.4.3 mRNA Integrity Check
1. Heat a water bath up to 65 °C. 2. Mix 0.8 g GIBCO agarose with 68.8 mL distilled water, note weight on Erlenmeyer flask. 3. Boil in microwave until agarose dissolves and readjust the weight with distilled water. 4. Cool solution to about 65 °C in the water bath. 5. Add 8 mL 10× MOPS (3-(N-morpholino)propanesulfonic acid). 6. Add 3.2 mL 37 % (12.3 M) formaldehyde and swirl gently (work in a hood). 7. Immediately pour the gel into the gel trays, put the combs in place and remove air bubbles. 8. Leave the gel for about 20–25 min (it has to appear slight milky) to let it become viscous. 9. Mix RNA samples with Northern Max® formaldehyde loading dye. Use between 500 and 1,000 ng RNA for gel electrophoresis. 10. Place the samples on ice. 11. Vortex the samples. 12. Incubate the samples for 15 min at 65 °C. 13. Place the samples on ice for 2 min.
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14. Vortex the samples shortly and spin them shortly afterwards. 15. Place the solidified gel in the electrophoresis tank. 16. Pour 1× MOPS buffer into the electrophoresis tank. The buffer should reach the top of the gel. Do not cover the gel with buffer solution. Remove the combs. 17. Fill the wells/slots with buffer. 18. Load the samples (8 μL per well/slot). 19. Run the electrophoresis at 100 V until the bromophenol blue has migrated 4.5 cm into the gel (50–65 min). 20. Prepare the HDGreen Plus DNA stain solution: 30 μL SYBR Green + 300 mL distilled water in a bowl; place the bowl on the shaker/tilt table. 21. Put off the mean, take out the gel tray and gently push the gel out off the gel tray on a plastic foil. 22. Remove the small plastic foils from the gel and then put the gel into the bowl with HDGreen Plus DNA stain solution. 23. Incubate the gel(s) for about 30 min in the HDGreen Plus DNA stain solution. 24. Take pictures of the RNA gel and examine 18S/28S bands (around 4.5 kb and 1.5 kb, respectively) in order to evaluate the RNA integrity. 3.4.4 Quantitative Polymerase Chain Reaction
1. Use sterile 96-well RT-PCR plates. 2. Cover all lanes of the 96-well plate with specific lids in order to protect the wells from any dust etc. 3. Pipette the respective volume of QuantiTect SYBR Green mastermix into each well and finish this step for all used plates, if you plan to measure several plates. Afterwards store the plates that are not directly measured in the fridge until further progress. Proceed only with one plate at a time (see Note 6). 4. Pipette the respective volume of your samples or standards into the respective well (see Note 7). 5. Afterwards, fix the lids on the plate, check whether the lids are located correctly (see Note 8). 6. Before placing the 96-well plate into the cycler, spin the plate by means of a small PCR plate centrifuge (see Note 9). 7. Place the 96-well PCR plate carefully in the cycler. 8. Run the respective PCR program (see Note 10). 9. Optionally, a hot start procedure can be applied. The hot start PCR is a good method to avoid nonspecific amplifications of DNA of which very low remaining stock can be caught in the RNA sample after purification from the tissue.
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Notes 1. In order to assess the optimal protein amount loaded per slot, perform a dilution series loading, e.g., 5, 10, 15, 20, 30, 40, 60, and 80 μg of total protein. Plot the optical density measured for the respective band against the respective total protein amount. The resulting plot should give a linear correlation between the optical density and the total protein amount. The optimal total protein amount loaded has to be determined for each protein of interest, before performing the sodium polyacrylamide gel electrophoresis and western blotting (see below), in order to perform establish optimal setting to reach the optimal result. 2. Some blotting systems (e.g., Trans-Blot Turbo system, BioRad) offer predefined blotting programs for different applications. Other blotting systems may not provide such programs; in either case, the optimal blotting setting (blotting time, current, buffer) has to be determined for each protein of interest to establish an optimal transfer system to obtain optimal results. 3. For each protein of interest the optimal blocking procedure has to be tested before the experiments. Usually, 5 % milk powder or 5 % bovine serum albumin dissolved in TBST is a good starting point. 4. If the isolated RNA is of pour quality and quantity, perform the additional steps after step 15 and before step 16, because this may improve the quality of RNA isolation due to repeated precipitation of the RNA from the solution in almost ultrapure EtOH. Due to the repeated precipitation of the RNA pellet, the purity of the RNA can be improved; however, one has to keep in mind that repeated RNA precipitation my result in lower RNA concentrations, a side effect that can be accepted when the quality of RNA is improved. Add vol(RNase-free water) 1:10 of vol(TriReagent) on the top of the pellet. Leave at RT for 5–10 min. Vortex to dissolve the pellet completely, spin shortly. Add vol(3 M NaAc, pH 5.5 (RNase-free)) 1:100 of vol(TriReagent) and mix (vortex), spin shortly. Add vol(96 % EtOH (RNase-free)) 1:5 of vol(TriReagent). My by inversion. Leave at RT for 2–15 min. Spin at 12,000 × g for 8 min at 4 °C. Remove the supernatant, about 310 μL. Add equal vol(75 % EtOH (RNase free)) to vol(TriReagent). Wash sides and lid by inversion. Spin at 7,500 × g for 5 min at 4 °C. Remove the supernatant, about 1,000 μL. Spin at 7,500 × g for 1 min at 4 °C. Remove the remaining supernatant, about 10 μL. 5. To remove remaining stocks of DNA, although at very low levels when the purification procedure was performed sufficiently, a DNA digestion procedure can be added.
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6. The user has to define by preliminary studies, which mastermix volume is sufficient for running the quantitative polymerase chain reaction. Usually, 25 or 50 μL are used. 7. The user has to define by preliminary studies, which sample or standard volume is sufficient for running the quantitative polymerase chain reaction. Usually, 5 μL are sufficient. 8. The correctly fixed lids are important as the light in the cycler will pass orthogonally. 9. This spinning step is important, since only small amounts of the mastermix-samples/standard mix at the tube edge can have significant influences on the measured results. 10. If it is planned to measure more than one PCR plate, the respective plates can be prepared until they are readily prepared for measuring in the PCR cycler. Afterwards, the readyto-use plates can be stored at −20 °C until further processing. Annealing temperature (Ta) for each primer pair (forward, reverse) has to be determined individually in order to guarantee the optimal quantitative polymerase chain reaction results. In general, a good starting point to determine the optimal annealing temperature is about 5 °C below the calculated melting temperature (Tm) [11]. References 1. Fluck M, Hoppeler H (2003) Molecular basis of skeletal muscle plasticity–from gene to form and function. Rev Physiol Biochem Pharmacol 146:159–216 2. Bentzinger CF, Wang YX, Dumont NA, Rudnicki MA (2013) Cellular dynamics in the muscle satellite cell niche. EMBO Rep 14:1062–1072 3. Mauro A (1961) Satellite cell of skeletal muscle fibers. J Biophys Biochem Cytol 9:493–495 4. Kuang S, Kuroda K, Le GF, Rudnicki MA (2007) Asymmetric self-renewal and commitment of satellite stem cells in muscle. Cell 129: 999–1010 5. Hochreiter-Hufford AE, Lee CS, Kinchen JM, Sokolowski JD, Arandjelovic S, Call JA, Klibanov AL, Yan Z, Mandell JW, Ravichandran KS (2013) Phosphatidylserine receptor BAI1 and apoptotic cells as new promoters of myoblast fusion. Nature 497:263–267 6. Abmayr SM, Pavlath GK (2012) Myoblast fusion: lessons from flies and mice. Development 139:641–656
7. Larsson LI, Bjerregaard B, Talts JF (2008) Cell fusions in mammals. Histochem Cell Biol 129: 551–561 8. Dupressoir A, Vernochet C, Bawa O, Harper F, Pierron G, Opolon P, Heidmann T (2009) Syncytin-A knockout mice demonstrate the critical role in placentation of a fusogenic, endogenous retrovirus-derived, envelope gene. Proc Natl Acad Sci U S A 106:12127–12132 9. Tsujita Y, Muraski J, Shiraishi I, Kato T, Kajstura J, Anversa P, Sussman MA (2006) Nuclear targeting of Akt antagonizes aspects of cardiomyocyte hypertrophy. Proc Natl Acad Sci U S A 103:11946–11951 10. Willkomm L, Schubert S, Jung R, Elsen M, Borde J, Gehlert S, Suhr F, Bloch W (2014) Lactate regulates myogenesis in C2C12 myoblasts in vitro. Stem Cell Res 12:742–753 11. Rychlik W, Spencer WJ, Rhoads RE (1990) Optimization of the annealing temperature for DNA amplification in vitro. Nucleic Acids Res 18:6409–6412
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Introduction Mammalian skeletal muscle tissue owns a remarkable ability to regenerate upon damage and thus shows a highly plastic morphology [1]. The regenerative capacity of mammalian skeletal muscle tissue is predominantly driven by a myogenic progenitor cell population known as satellite cells [2]. Myogenic progenitors are interposed between the basal lamina and the sarcolemma of adult muscle fibers [3]. Upon tissue damage, e.g., after exhaustive mechanical exercise or during muscle disease, these myogenic progenitors are able to become activated and to enter the cell proliferation cycle, to divide (asymmetrically), to differentiate, and to fuse with adjacent muscle fibers [4]. Therefore, intercellular
Kurt Pfannkuche (ed.), Cell Fusion: Overviews and Methods, Methods in Molecular Biology, vol. 1313, DOI 10.1007/978-1-4939-2703-6_8, © Springer Science+Business Media New York 2015
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fusions among mononucleated myogenic progenitors with each other and among myogenic progenitors with multinucleated adult muscle fibers are required for the development and specifically for the maintenance of intact and integral adult muscle fibers [5, 6]. The formation of syncytia thus is an integral process to maintain functional muscle fibers in the adult mammalian organism. Different methodological approaches can be used to study fusion events in mammalian skeletal muscle tissue. Microscopic approaches, such as confocal microscopy, have the advantage to visualize ongoing fusion events in cell cultures or in adult skeletal muscle tissues allowing for the identification of the exact localization of the event, while a quantification of ongoing fusion events is impossible. Cell fusion events are complex and concerted events making a systemic evaluation in adult skeletal muscle tissue difficult, but not impossible. In order to produce as clear ideas as possible about cell fusion events, a combination of in vitro cell culture systems and ex vivo skeletal muscle tissue analyses should be preferred. Classical protein and mRNA analysis approaches, such as western blot and quantitative real-time polymerase chain reaction (qPCR), are perfect tools to quantify cellular fusion events in cell cultures, e.g., C2C12 cells. Amongst in vitro systems, adult skeletal muscle tissues isolated from rodents or humans can be harvested, e.g., after in vivo mechanical stimulation experiments inducing microdamage in skeletal muscles. Due to these impacts, skeletal muscle tissues are susceptible for the induction of cell fusion events, why microscopic, western blot and qPCR analyses are promising tools to evaluate the levels of markers reflecting cell fusion events present in skeletal muscle tissue. However, using these approaches, it is not possible to exactly localize the fusion events, why high-resolution confocal microscopy provides the power to precisely localize the spots of cell fusion events. Fusion events in mammalian skeletal muscle tissue have not been a broad focus in the literature, yet, why the knowledge about classical fusion markers is still limited; however, there are some proteins known to be involved in fusion events in skeletal muscle tissue [6, 7]. Among these proteins, the family of syncytin proteins, mostly known as critical players in placentogenesis [8], seems to be involved in cellular fusion initiated in adult skeletal muscle tissue. These markers and also related ones can be assessed by microscopic and western blot/qPCR methods in order to investigate cell fusion events in adult skeletal muscle tissue. Herein, I will describe methodological approaches to study fusion events in mammalian skeletal muscle tissue. I will describe syncytin-1 analysis in both cell culture of myogenic progenitors and skeletal muscle tissue from mice stressed in vivo by mechanical stimulation.
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Materials Prepare all solutions using ultrapure water and analytical grade reagents. Use only sterile plastic consumables. Autoclave solutions etc. before use.
2.1
Cell Culture
1. C2C12 cells. 2. Uncoated cell culture flasks. 3. C2C12 proliferation medium (500 mL): 370 mL DMEM, 100 mL 20 % fetal calf serum (FCS), 5 mL 1 % penicillin/ streptomycin (100× stock solution, Invitrogen Life Technologies), 10 mL 4 mM L-glutamine, 10 mL 1.5 g/L sodium bicarbonate, 5 mL 1 mM sodium pyruvate. 4. C2C12 differentiation medium (500 mL): 450 mL DMEM, 20 mL 4 % horse serum, 5 mL 1 % penicillin/streptomycin, 10 mL 4 mM L-glutamine, 10 mL 1.5 g/L sodium bicarbonate, 5 mL 1 mM sodium pyruvate. 5. Sterile cell culture bench. 6. Cell culture.
2.2 Confocal Microscopy
1. Tris-buffered saline (TBS): 0.05 M Tris-base, 0.15 M sodium chloride (NaCl), use 1 N hydrochloric acid to adjust pH to 7.6. 2. Phosphate-buffered saline (0.2 M PBS): 28.8 g water-free disodium hydrogen phosphate, 5.2 g sodium hydrogen phosphate-monohydrate, 17.5 g sodium chloride, pH 7.4. 3. 4 % PFA fixative: dissolve 4 g paraformaldehyde in 40 mL ultrapure water under heating (60 °C, avoid cooking), clear the solution by dropwise addition of 1 N hydrochloric acid, filtrate the solution afterwards, mix with 50 mL 0.2 M PBS, adjust pH to 7.4, add ultrapure water to 100 mL. 4. Cryo microtome (Leica CM 1900). 5. Microscope slides, poly-Lysine coated. 6. 3 % hydrogen peroxide solution in methanol: Mix 0.075 mL 30 % hydrogen peroxide solution with 0.675 mL ultrapure water and 3 mL methanol. 7. 0.5 M ammonium chloride-TBST solution: Dissolve 0.59 g ammonium chloride in 20 mL TBS and add 50 μL Triton-X 100. 8. 5 % bovine serum albumin solution (15 mL): Dissolve 0.75 g bovine serum albumin (BSA) in 15 mL ultrapure water. 9. 0.8 % bovine serum albumin solution (15 mL): Dissolve 0.12 g bovine serum albumin (BSA) in 15 mL ultrapure water. 10. Primary polyclonal syncytin-1 antibody produced in rabbit (Abnova).
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11. Secondary antibodies Alexa Fluor® 555 goat anti-rabbit (Invitrogen Life Technologies). 12. DNA staining dyes: 4′,6-Diamidino-2-Phenylindole dihydrochloride (DAPI) nuclear acid staining dye, NucRed® Live 647 Ready probes reagent (Draq5) nuclear acid staining dye (Invitrogen Life Technologies). 13. Aqua-Poly/Mount water-soluble, non-fluorescing mounting medium (Polysciences). 14. Confocal microscope (e.g., LSM 510Meta equipped with one NeHe laser generating a laser line with a wavelength 543 nm and one NeHe laser generating a laser line with a wavelength 633 nm). 2.3 Protein Isolation from Skeletal Muscle Tissue
1. 20–30 mg skeletal muscle tissue. 2. Liquid nitrogen (N2). 3. Mortar and pestle. 4. Cell lysis buffer (Cell Signaling). Add PMSF (1:100 v:v) and phosphatase inhibitor (1:50 v:v) to commercially available 1× cell lysis buffer. 5. 100 μM PMSF and 50 μm phosphatase inhibitor (Thermo Scientific). 6. SDS polyacrylamide gel electrophoresis: Sterile 1.5 mL Eppendorf tubes.
2.4 Protein Content Determination
1. Bradford protein assay kit.
2.5 SDS Polyacrylamide Gel Electrophoresis
1. SDS-PAGE running buffer: 0.025 M Tris-base, 0.192 M glycine, 10 % (m:v) sodium dodecylsulfate (SDS), pH 8.3. 2. 30 % acrylamide/Bis-acrylamide solution (37.5:1 acrylamide– bis). Commercially available ready-to-use solution. 3. 10 % sodium dodecylsulfate (SDS) solution: 10 g SDS dissolved in 100 mL ultrapure water, agitate very carefully to prevent excess foaming. 4. 10 % ammonium peroxodisulfate (APS) solution: 10 g dissolved in 100 mL ultrapure water. Store at 4 °C for no longer than 4 weeks. 5. Resolution gel buffer: 1.5 M Tris-base, pH 8.8. Store at room temperature. 6. Stacking gel buffer: 0.5 M Tris-base, pH 6.8. Store at room temperature. 7. Sample lysis buffer (2× Laemmli buffer): 0.125 M Tris-base, 4 % SDS, 10 % β-mercaptoethanol, 20 % glycerol, 0.004 % bromophenol blue, pH 6.8. Store aliquots frozen at −20 °C. 8. N,N,N′,N′-tetramethylethylenediamine (TEMED), commercial ready-to-use solution.
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2.6
Western Blot
1. Polyvinylidene fluoride (PVDF) membrane. 2. Tris-buffered saline (TBS, 10×, 2 L): 121.40 g Tris base, 175.32 g NaCl, adjust pH to 7.6 using HCl. 3. TBST: TBS containing 0.05 % Tween-20. 4. Blocking solution: 5 % milk powder in TBST. Prepare freshly immediately before use. 5. Primary and secondary antibody diluents solution: 5 % bovine serum albumin in TBST. 6. Mini PROTEAN® Bio-Rad).
tetra-cell
system
(1.5
mm
spacer,
7. Trans-Blot Turbo system (Bio-Rad). 8. Western blot transfer buffer (Towbin buffer): 0.025 M Trisbase, 0.192 M glycine, pH 8.3. Immediately before use add 5–20 % (v:v) methanol. 9. Protein standard ladder. 10. Whatman blotting paper. 11. Primary polyclonal syncytin-1 antibody produced in rabbit (Abnova). 12. Primary monoclonal glyceraldehyde-3-phosphate dehydrogenase (Gapdh) antibody raised in rabbit (Cell Signaling). Gapdh can be used as a “housekeeping” protein. 13. Secondary antibody: Stabilized goat anti-rabbit IgG (H + L), peroxidase-conjugated (Thermo Scientific). 14. Enhanced chemiluminescence (ECL) western blotting substrate kit. 2.7 mRNA Isolation from Skeletal Muscle
1. 20–30 mg skeletal muscle tissue. 2. Liquid nitrogen (N2). 3. Mortar and pestle. 4. Sterile 2.0 mL tubes. 5. TriReagent 6. 1-bromo-3-chloropropane. 7. Ethanol, molecular grade. 8. 3 M Sodium acetate, adjust pH to 5.5 by glacial acetic acid. 9. RNase-free water. 10. Agarose. 11. HDGreen Plus DNA stain to visualize RNA (Intas).
2.8 Quantitative Polymerase Chain Reaction (qPCR)
1. Omniscript RT Kit for cDNA synthesis (Qiagen). 2. Barrier Tips 1,000, 200, 100, 10 μL (low binding tips). 3. DNA low binding tubes 0.5 and 1.5 mL.
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4. PCR 96 tube plates. 5. Strips of eight PCR caps. 6. QuantiTect SYBR Green PCR Kit (Qiagen). 7. Syncytin-1 primers (forward: CAT CTA TGC TGG ATG AAG CCT, reverse: AGA CCC TGG CAT GGC CAT TA) [8]. 8. Gapdh primers (forward: GAC ATG CCG CCT GGA GAA AC, reverse: AGC CCA GGA TGC CCT TTA GT) [9]; Gapdh can be used as a “housekeeping” gene. 9. Thermocycler. 10. 10× MOPS (3-(N-morpholino)propanesulfonic acid): dissolve 41.8 g of MOPS in 700 mL ultrapure water, adjust pH to 7.0 with 2 N NaOH, add 20 mL 1 M sodium acetate and 20 mL of 0.5 M EDTA, adjust the volume to 1 L buffer and leave the flask in the water bath, mix the solution; to make 1× MOPS buffer dilute (100 mL 10× MOPS buffer in 900 mL ultrapure water). 11. Northern Max® formaldehyde loading dye (Life Technologies).
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Methods In the following, different methods are described to establish and run a C2C12 cell culture system in the lab for supportive experimental approaches for the study of cell fusion events in adult skeletal muscle tissue, to perform high quality confocal microscopy, to perform western blotting, and to perform quantitative polymerase chain reaction.
3.1
Cell Culture
1. Keep C2C12 cells in cell culture flasks at 37 °C and 5 % CO2 in proliferation medium. 2. For experimental procedures plate C2C12 cells on gelatinecoated (0.1 % in PBS) glass cover slips or petri dishes at a density of 10,000 cells/cm2. 3. Keep C2C12 cells in proliferation medium until 80–90 % confluence is reached. 4. Harvest proliferating C2C12 cells for further experiments (e.g., confocal microscopy, western blotting, quantitative polymerase chain reaction). 5. Switch medium to differentiation medium and maintain C2C12 up to 10 days in culture. 6. Change media every second day [10].
3.2 Confocal Microscopy
1. Store the skeletal muscle tissue to be analyzed for at least 20 min at −20 °C within the cryo microtome. 2. Cut 7 μm slices of the skeletal muscle tissue by means of the cryo microtome and mount the slices on the microscope slide.
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Place at least three consecutive slices on one microscope slide to enable the staining of positive slices and also negative controls. 3. Use a grease pencil to edge each single slice mounted on the microscope slide. This can be repeated several times when the edge is removed during incubation steps. 4. Wash the slices three times 5 min with TBS. 5. Block endogenous peroxidases. Incubate the slices with 3 % hydrogen peroxide solution for 10 min. 6. Wash the slices three times 5 min with TBS. 7. Permeabilize the skeletal muscle sarcolemma and unmask the epitopes by incubation of the slices with Triton-X 100 solution. 8. Wash the slices three times 5 min with TBS. 9. Blocking of unspecific binding epitopes with 5 % bovine serum albumin solution. 10. Incubate the slices with the primary antibody against syncytin1 (dilution 1:100) dissolved in 0.8 % bovine serum albumin solution. Incubate for at least 16 h overnight at 4 °C. 11. Use one slice on each microscope slide as negative control. Incubate this slice with 0.8 % bovine serum albumin without the primary antibody against syncytin-1 (End of day 1). 12. Wash the slices three times 5 min with TBS. 13. Incubate all slices with the secondary antibody Alexa Fluor® 555 goat anti-rabbit (dilution 1:500) dissolved in TBS solution for at least 60 min at room temperature. 14. Wash the slices three times 5 min with TBS. 15. Incubate all slices with DNA staining dyes: 4′,6-Diamidino-2Phenylindole, dihydrochloride (Dapi) nuclear acid staining dye (dilution 1:10,000) and NucRed® Live 647 Ready probes reagent (Draq5) nuclear acid staining dye (dilution in 1:10,000) dissolved in TBS solution. 16. Mount microscope slides with Aqua-Poly/Mount mounting medium. 17. Evaluate the slices by means of a confocal microscope (Fig. 1). 3.3 SDS Polyacrylamide Gel Electrophoresis/ Western Blot 3.3.1 Tissue Homogenization and Sample Preparation
1. Prepare 1.5 mL tubes containing 200 μL 1× cell lysis buffer; name the tube. 2. Cool the tubes on crushed ice. 3. Use mortar and pestle to homogenize skeletal muscle tissue to a fine powder in liquid nitrogen (N2). 4. Transfer skeletal muscle powder carefully to the prepared 1× cell lysis buffer-containing tube.
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Fig. 1 Syncytin-1 immunofluorescence staining in skeletal muscle tissue. (a) Syncytin-1 (stained by means of Alexa 555) is expressed at low levels at sedentary conditions. (b) After 6 weeks to chronic exercise, syncytin-1 is pronouncedly increased in skeletal muscle tissue (Tissue from Suhr et al., submitted). Bar = 100 μm
5. Vortex at least for 20 s to dissolve the powdered skeletal muscle tissue slowly in lysis buffer. 6. Spin shortly to remove cell lysis buffer from the lid of the tubes. 7. Vortex roughly for 30 s. 8. Spin at 9,000 × g for 5 min at 4 °C. 9. Carefully transfer the supernatant into a freshly prepared 1.5 mL tube. 10. Spin supernatant at 9,000 × g for 20 min at 4 °C. 11. Carefully transfer the supernatant into a freshly prepared 1.5 mL tube. 12. Measure protein concentration by means of the Bio-Rad Bradford protein assay kit. 13. Dilute samples with 2× Laemmli buffer to the respective needed protein concentration. Afterwards, heat the lysate/ Laemmli buffer mixture up to 95 °C for 5 min. 14. Let the samples cool down to room temperature and centrifuge the samples to remove condensates from the lid and the edge of the tubes. 15. Store samples on ice when they are processed immediately or store at −80 °C until usage.
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1. Handle the Mini PROTEAN® tetra-cell system as instructed by the manual, use 1.5 mm spacer. 2. Pour a 10 % acrylamide-containing gel (10 mL solution): 3.3 mL ultrapure water, 2.7 mL 30 % acrylamide solution, 2.5 mL resolution gel buffer, 100 μL 10 % SDS, 10 % APS, 5 μL TEMED. 3. Cover the gel with 500 μL isopropanol. 4. Let the gel polymerize for at least 30 min at room temperature. 5. In between pour a 5 % stacking gel (5 mL solution): 3.40 mL ultrapure water, 0.83 mL 30 % acrylamide solution, 0.63 mL resolution gel buffer, 50 μL 10 % SDS, 50 μL 10 % APS, 5 μL TEMED. 6. Remove the isopropanol from the resolution gel and pour the stacking gel on top of the resolution gel. 7. Place the comb immediately on top of the freshly poured stacking gel. 8. Let the gel polymerize for at least 30 min at room temperature. 9. Place the gel-containing tray into the tank and fill the gel electrophoresis buffer into both the tank and the gel-containing tray. 10. Carefully remove the comb from the stacking gel and carefully rinse the slots with gel electrophoresis buffer using a Pasteur pipette. 11. Carefully load protein marker standards into the respective slot. 12. Carefully load the samples into the respective slot. Usually, 15–20 mg of total protein is sufficient for gel electrophoresis coupled to western blot procedures (see Note 1). 13. Start running the gel at constant 80 V to allow samples to accumulate in the stacking and to transmigrate into the resolution gel (takes around 15 min). Afterwards increase the electrical potential to 140 V in order to transmigrate the samples through the resolution gel. 14. Stop running the gel when the resolution of the gel fits with the experimental setup (takes around 45–60 min), e.g., optimal resolution range for the detection of syncytin-1 or other proteins of interest. 15. Take out the gel-containing tray from the tank and process the gels further for western blot (see below).
3.3.3 Western Blot
1. Soak Whatman papers in freshly prepared 1× transfer buffer (Towbin buffer).
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2. Activate PVDF membrane in 100 % methanol for 10 s, afterwards deposit PVDF membrane in freshly prepared 1× transfer buffer (Towbin buffer). 3. Prepare the semidry blotting apparatus (e.g., Trans-Blot Turbo system, Bio-Rad) as indicated in the manual. Arrange Whatman papers, PVDF membrane and polyacrylamide gel as follows (from bottom, cathode, to top, anode): three Whatman papers, PVDF membrane, polyacrylamide gel, two Whatman papers. When stacking the Whatman papers, PVDF membrane and polyacrylamide gel on top of each other as indicated, make sure that no air bubbles remain between individual layers. Otherwise a constant current field cannot pass the layers resulting in lower signal detection. 4. Run the semidry blotting apparatus (e.g., Trans-Blot Turbo system, Bio-Rad) as indicated in the manual for middle-large proteins (see Note 2). 5. After finishing the run of the semidry blotting apparatus (e.g., Trans-Blot Turbo system, Bio-Rad) cut PVDF membrane in two parts: one part to enable for example syncytin-1 detection (around 55 kDa) and one part to enable Gapdh detection (around 36 kDa). 6. Incubate PVDF membranes in 5 % milk powder blocking solution for at least 90 min at room temperature. The standard protein ladder should be visible on PVDF membranes (see Note 3). 7. Prepare primary antibody solutions: dissolve the primary antibody against syncytin-1 raised in rabbit in 5 % bovine serum albumin solution (dilution 1:200); dissolve the primary antibody against Gapdh raised in rabbit in 5 % bovine serum albumin solution (dilution 1:15,000). 8. Incubate/hybridize PVDF membranes for at least 16 h overnight at 4 °C with the respective diluted primary antibody solution. 9. Wash with TBST solution at least three times 10 min at room temperature. 10. Incubate/hybridize PVDF membranes with secondary antibody solutions: dissolve secondary antibody stabilized goat anti-rabbit IgG (H + L), peroxidase-conjugated (dilution 1:2,000) in TBST and incubate PVDF membranes for at least 60 min at room temperature. 11. Wash with TBST solution at least three times 10 min at room temperature. 12. Incubate/hybridize PVDF membranes with enhanced chemiluminescence (ECL) western blotting substrate kit for 2–3 min. 13. Visualize PVDF membranes using either an imaging system (e.g., Chemidoc, Bio-Rad) or X-ray films in a dark room (Fig. 2).
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Fig. 2 Syncytin-1 expression measured by western blot in skeletal muscle tissue. Syncytin-1 is expressed at low levels at sedentary (Sed) conditions. After 6 weeks to chronic exercise (Exerc), syncytin-1 is pronouncedly increased in skeletal muscle tissue. These data correspond to the data shown in Fig. 1. Gapdh was used as a “housekeeping” protein (Tissue from Suhr et al., submitted)
3.4 Quantitative Polymerase Chain Reaction
1. Prepare 2 mL tubes containing 1,000 μL TriReagent, name the tube.
3.4.1 RNA Isolation: Tissue Homogenization
3. Use mortar and pestle to homogenize skeletal muscle tissue to a fine powder in liquid nitrogen (N2).
2. Cool the tubes on crushed ice.
4. Transfer skeletal muscle powder carefully to the prepared TriReagent-containing tube. 3.4.2 RNA Isolation: Purification
1. Spin shortly to remove TriReagent from the lid of the tubes. 2. Add the volume (BCP) to the TriReagent solution in a 1:10 ratio. 3. Vortex tubes for 15 s. 4. Leave at RT for 2–15 min. 5. Spin at 12,000 × g for 15 min at 4 °C. The solution separates: upper aqueous phase (RNA), interphase (DNA), lower red phase (proteins and RNases). 6. Transfer most of the aqueous phase (450 μL) to a fresh 1.5 mL tube. Avoid contact with interphase and lower phase. In case of contact, spin again. Interphase and organic phase may be stored at −80 °C for further purifications. 7. Add an exactly equal volume of isopropanol to the aqueous phase to precipitate the RNA. Mix by inversion. 8. Leave at RT for 5–10 min. 9. Spin at 12,000 × g for 8 min at 4 °C. Place the tube upwards. The pellet should be visible after spinning.
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10. Remove the supernatant (about 900 μL). 11. Add an equal volume of 75 % EtOH (RNase free) per volume (TriReagent). Wash sides and lid by inversion. 12. Spin at 7,500 × g for 5 min at 4 °C. 13. Remove the supernatant. 14. Spin at 7,500 × g for 1 min at 4 °C. 15. Remove the remaining supernatant, about 10 μL (see Notes 4 and 5). 16. Air-dry the pellet for 5 min, but do not dry the pellet completely as it will be difficult to get resuspended. 17. Dissolve the pellet in RNase-free water. Vortex and spin shortly at the end. 18. Measure RNA concentration and purity by means of for example NanoDrop machine (Thermo Scientific) following the manuals for RNA measurement. 19. RNA concentration should yield about 200–1,000 ng/mL. 20. 260/280 ratio should be 1.8–2.0, 260/230 ratio should be 2.0–2.2 in order to guarantee high RNA integrity and purity (for details see manuals of the NanoDrop machine). 3.4.3 mRNA Integrity Check
1. Heat a water bath up to 65 °C. 2. Mix 0.8 g GIBCO agarose with 68.8 mL distilled water, note weight on Erlenmeyer flask. 3. Boil in microwave until agarose dissolves and readjust the weight with distilled water. 4. Cool solution to about 65 °C in the water bath. 5. Add 8 mL 10× MOPS (3-(N-morpholino)propanesulfonic acid). 6. Add 3.2 mL 37 % (12.3 M) formaldehyde and swirl gently (work in a hood). 7. Immediately pour the gel into the gel trays, put the combs in place and remove air bubbles. 8. Leave the gel for about 20–25 min (it has to appear slight milky) to let it become viscous. 9. Mix RNA samples with Northern Max® formaldehyde loading dye. Use between 500 and 1,000 ng RNA for gel electrophoresis. 10. Place the samples on ice. 11. Vortex the samples. 12. Incubate the samples for 15 min at 65 °C. 13. Place the samples on ice for 2 min.
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14. Vortex the samples shortly and spin them shortly afterwards. 15. Place the solidified gel in the electrophoresis tank. 16. Pour 1× MOPS buffer into the electrophoresis tank. The buffer should reach the top of the gel. Do not cover the gel with buffer solution. Remove the combs. 17. Fill the wells/slots with buffer. 18. Load the samples (8 μL per well/slot). 19. Run the electrophoresis at 100 V until the bromophenol blue has migrated 4.5 cm into the gel (50–65 min). 20. Prepare the HDGreen Plus DNA stain solution: 30 μL SYBR Green + 300 mL distilled water in a bowl; place the bowl on the shaker/tilt table. 21. Put off the mean, take out the gel tray and gently push the gel out off the gel tray on a plastic foil. 22. Remove the small plastic foils from the gel and then put the gel into the bowl with HDGreen Plus DNA stain solution. 23. Incubate the gel(s) for about 30 min in the HDGreen Plus DNA stain solution. 24. Take pictures of the RNA gel and examine 18S/28S bands (around 4.5 kb and 1.5 kb, respectively) in order to evaluate the RNA integrity. 3.4.4 Quantitative Polymerase Chain Reaction
1. Use sterile 96-well RT-PCR plates. 2. Cover all lanes of the 96-well plate with specific lids in order to protect the wells from any dust etc. 3. Pipette the respective volume of QuantiTect SYBR Green mastermix into each well and finish this step for all used plates, if you plan to measure several plates. Afterwards store the plates that are not directly measured in the fridge until further progress. Proceed only with one plate at a time (see Note 6). 4. Pipette the respective volume of your samples or standards into the respective well (see Note 7). 5. Afterwards, fix the lids on the plate, check whether the lids are located correctly (see Note 8). 6. Before placing the 96-well plate into the cycler, spin the plate by means of a small PCR plate centrifuge (see Note 9). 7. Place the 96-well PCR plate carefully in the cycler. 8. Run the respective PCR program (see Note 10). 9. Optionally, a hot start procedure can be applied. The hot start PCR is a good method to avoid nonspecific amplifications of DNA of which very low remaining stock can be caught in the RNA sample after purification from the tissue.
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Notes 1. In order to assess the optimal protein amount loaded per slot, perform a dilution series loading, e.g., 5, 10, 15, 20, 30, 40, 60, and 80 μg of total protein. Plot the optical density measured for the respective band against the respective total protein amount. The resulting plot should give a linear correlation between the optical density and the total protein amount. The optimal total protein amount loaded has to be determined for each protein of interest, before performing the sodium polyacrylamide gel electrophoresis and western blotting (see below), in order to perform establish optimal setting to reach the optimal result. 2. Some blotting systems (e.g., Trans-Blot Turbo system, BioRad) offer predefined blotting programs for different applications. Other blotting systems may not provide such programs; in either case, the optimal blotting setting (blotting time, current, buffer) has to be determined for each protein of interest to establish an optimal transfer system to obtain optimal results. 3. For each protein of interest the optimal blocking procedure has to be tested before the experiments. Usually, 5 % milk powder or 5 % bovine serum albumin dissolved in TBST is a good starting point. 4. If the isolated RNA is of pour quality and quantity, perform the additional steps after step 15 and before step 16, because this may improve the quality of RNA isolation due to repeated precipitation of the RNA from the solution in almost ultrapure EtOH. Due to the repeated precipitation of the RNA pellet, the purity of the RNA can be improved; however, one has to keep in mind that repeated RNA precipitation my result in lower RNA concentrations, a side effect that can be accepted when the quality of RNA is improved. Add vol(RNase-free water) 1:10 of vol(TriReagent) on the top of the pellet. Leave at RT for 5–10 min. Vortex to dissolve the pellet completely, spin shortly. Add vol(3 M NaAc, pH 5.5 (RNase-free)) 1:100 of vol(TriReagent) and mix (vortex), spin shortly. Add vol(96 % EtOH (RNase-free)) 1:5 of vol(TriReagent). My by inversion. Leave at RT for 2–15 min. Spin at 12,000 × g for 8 min at 4 °C. Remove the supernatant, about 310 μL. Add equal vol(75 % EtOH (RNase free)) to vol(TriReagent). Wash sides and lid by inversion. Spin at 7,500 × g for 5 min at 4 °C. Remove the supernatant, about 1,000 μL. Spin at 7,500 × g for 1 min at 4 °C. Remove the remaining supernatant, about 10 μL. 5. To remove remaining stocks of DNA, although at very low levels when the purification procedure was performed sufficiently, a DNA digestion procedure can be added.
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6. The user has to define by preliminary studies, which mastermix volume is sufficient for running the quantitative polymerase chain reaction. Usually, 25 or 50 μL are used. 7. The user has to define by preliminary studies, which sample or standard volume is sufficient for running the quantitative polymerase chain reaction. Usually, 5 μL are sufficient. 8. The correctly fixed lids are important as the light in the cycler will pass orthogonally. 9. This spinning step is important, since only small amounts of the mastermix-samples/standard mix at the tube edge can have significant influences on the measured results. 10. If it is planned to measure more than one PCR plate, the respective plates can be prepared until they are readily prepared for measuring in the PCR cycler. Afterwards, the readyto-use plates can be stored at −20 °C until further processing. Annealing temperature (Ta) for each primer pair (forward, reverse) has to be determined individually in order to guarantee the optimal quantitative polymerase chain reaction results. In general, a good starting point to determine the optimal annealing temperature is about 5 °C below the calculated melting temperature (Tm) [11]. References 1. Fluck M, Hoppeler H (2003) Molecular basis of skeletal muscle plasticity–from gene to form and function. Rev Physiol Biochem Pharmacol 146:159–216 2. Bentzinger CF, Wang YX, Dumont NA, Rudnicki MA (2013) Cellular dynamics in the muscle satellite cell niche. EMBO Rep 14:1062–1072 3. Mauro A (1961) Satellite cell of skeletal muscle fibers. J Biophys Biochem Cytol 9:493–495 4. Kuang S, Kuroda K, Le GF, Rudnicki MA (2007) Asymmetric self-renewal and commitment of satellite stem cells in muscle. Cell 129: 999–1010 5. Hochreiter-Hufford AE, Lee CS, Kinchen JM, Sokolowski JD, Arandjelovic S, Call JA, Klibanov AL, Yan Z, Mandell JW, Ravichandran KS (2013) Phosphatidylserine receptor BAI1 and apoptotic cells as new promoters of myoblast fusion. Nature 497:263–267 6. Abmayr SM, Pavlath GK (2012) Myoblast fusion: lessons from flies and mice. Development 139:641–656
7. Larsson LI, Bjerregaard B, Talts JF (2008) Cell fusions in mammals. Histochem Cell Biol 129: 551–561 8. Dupressoir A, Vernochet C, Bawa O, Harper F, Pierron G, Opolon P, Heidmann T (2009) Syncytin-A knockout mice demonstrate the critical role in placentation of a fusogenic, endogenous retrovirus-derived, envelope gene. Proc Natl Acad Sci U S A 106:12127–12132 9. Tsujita Y, Muraski J, Shiraishi I, Kato T, Kajstura J, Anversa P, Sussman MA (2006) Nuclear targeting of Akt antagonizes aspects of cardiomyocyte hypertrophy. Proc Natl Acad Sci U S A 103:11946–11951 10. Willkomm L, Schubert S, Jung R, Elsen M, Borde J, Gehlert S, Suhr F, Bloch W (2014) Lactate regulates myogenesis in C2C12 myoblasts in vitro. Stem Cell Res 12:742–753 11. Rychlik W, Spencer WJ, Rhoads RE (1990) Optimization of the annealing temperature for DNA amplification in vitro. Nucleic Acids Res 18:6409–6412
