Methods in Molecular Biology DOI 10.1007/7651_2014_68 © Springer Science+Business Media New York 2014
Analysis of Intracellular Calcium Signaling in Human Embryonic Stem Cells Adrienn Pe´ntek, Katalin Pa´szty, and A´gota Apa´ti Abstract Measurement of changes in intracellular calcium concentration is one of the most common and useful tools for studying signal transduction pathways or cellular responses in basic research and drug screening purposes as well. Increasing number of such applications using human pluripotent stem cells and their derivatives requires development of calcium signal measurements for this special cell type. Here we describe a modified protocol for analysis of calcium signaling events in human embryonic stem cells, which can be used for other pluripotent cell types (such as iPSC) or their differentiated offspring as well. Keywords: Human embryonic stem cell, Calcium signaling, Fluorescent calcium indicators, Genetically encoded calcium indicators, Confocal microscopy
1
Introduction Calcium signals control diverse cellular processes, for example regulation of gene expression (1, 2), cell division or apoptosis (3), and activation of excitatory cell types (4). Analysis of these versatile patterns of calcium signals is essential for studying the mechanisms lying beyond the cellular functions. There are generally two widely used classes of calcium indicators, chemical calcium indicators (5, 6) and genetically encoded calcium indicators (GECIs) (7–9) for calcium imaging. Both types of indicators exhibit altered fluorescent properties according to changes in cytoplasmic calcium concentration. Selection of proper calcium indicator depends on the amplitude, frequency, and spatial and temporal properties of the Ca2+ transient; however, the “global” Ca2+ signals can be measured by most of the commonly used calcium indicators (10, 11). Human embryonic stem cell lines have normal genetic background and can differentiate toward various cell types which make them a favorable model system for studying calcium signals evoked by endogenous substrates and a wide range of drugs. At the same time there are only limited numbers of publications about calcium signals in human pluripotent stem cells (12, 13). This is partly due to the special feature of these cell types such as the formation of
Adrienn Pe´ntek et al.
three-dimensional clumps, presence of feeder cells, sensitivity of human PSCs to any enzymatic or mechanical cell separation, and heterogeneous shape and function of the PSCs even in a given clump (cell aggregate). Here we present a commonly usable method based on confocal microscopic system which eliminates the abovementioned problems and can be used for calcium signal analysis of hESC and hiPSC lines too.
2
Materials
2.1 Maintenance of hES Cells
All media were sterile filtered with 0.22 μm Steritop-GP (Millipore) vacuum filter units. All media and reagents were added at room temperature. 1. 0.1 % gelatin solution in water (see Note 1). 2. Feeder cell layer: Mouse embryonic fibroblast (MEF) cells isolated from embryos at day 13 (Millipore) and treated by Mitomycin C (Sigma). 3. MEF culturing medium: 90 % Dulbecco’s Modified Eagle Medium (DMEM, Gibco) supplemented with 10 % Fetal Bovine Serum (Gibco), 1 mM Glutamax-I (Gibco). Store at 4 C. The medium may be used within 2 weeks of preparation. 4. hES culturing medium: 80 % Knockout Dulbecco’s Modified Eagle Medium (KoDMEM, Gibco) supplemented with 15 % Knockout Serum Replacement (Gibco), 1 mM Glutamax-I (Gibco), 0.1 mM beta-mercaptoethanol, 1 % nonessential amino acids, and 4 ng/mL human fibroblast growth factor (Invitrogen). Store at 4 C. The medium may be used within 2 weeks of preparation. 5. hES cells were propagated on mitomycin C-treated MEF feeder layer by enzymatic dissociation with 0.05 % trypsin–EDTA (Invitrogen) and replated on fresh feeder layer every second day as described earlier (14). 6. For confocal microscopic measurements cells were seeded onto an eight-well Nunc Lab-Tek II Chambered Coverglass (Nalgene Nunc International).
2.2 Calcium Signal Analysis in hES Cells
1. Fluo4-AM: 1 mM solution in dimethyl sulfoxide (DMSO) (see Note 2). 2. Hank’s balanced salt solution (HBSS) with calcium and magnesium, without phenol red (Invitrogen). Store at room temperature. 3. 100 mM adenosine-50 -triphosphate (ATP) solution (GE Life Sciences) (see Note 3).
Analysis of Calcium Signaling
4. Ionomycin (Invitrogen): 1 mM solution in DMSO (Invitrogen). Store at 20 C. Ionomycin was used at a 5 μM final concentration for measurements. 5. EGTA: 100 mM solution in water, pH 7.4 (see Note 4).
3
Methods
3.1 Loading of hESCs with the Calcium Indicator, Fluo4-AM
1. Cover the eight-well chambered cover glass with 250 μL of 0.1 % gelatin solution for each well. 2. Store the coated chamber at room temperature under laminar flow cabinet for 2 h. 3. Remove the remaining gelatin solution, and seed mitomycin C-treated MEF cells onto the wells (4 104 cell/well in 250 μL MEF culturing medium). Prepare the MEF feeder layer at least 1 day before plating the hESCs (see Note 5). 4. Seed hESC cells (20–40 clumps/well) on to the mitomycin C-treated MEF feeder layer in 500 μL hES culturing medium 24–48 h prior to calcium signal measurement (see Note 6). 5. On the day of experiment dilute 0.3 μL of 1 mM Fluo4-AM in 300 μL FBS-free KoDMEM (1 μM final concentration). Keep this solution at room temperature in the dark (see Note 7). 6. Remove the hES culturing medium, add 200 μL diluted Fluo4AM dye solution to one well, and keep the chamber in a CO2 incubator for 15–30 min. 7. Wash the cells with HBSS before confocal imaging. 8. Finally, add 200 μL of HBSS solution to the cells and place the chamber onto the microscope (see Note 8).
3.2 Microscope Settings
1. All settings are adjusted to an Olympus IX-81 laser scanning confocal microscope using an Olympus PLAPO 20 objective. For Fluo4 imaging excite cells at 488 nm and collect emission data between 505 and 535 nm. Set image resolution to 512 512 or lower and scan speed to fast (about 1 s/scan) so that time resolution might be around 1 s. z-resolution should be 4 μm, and the duration of the measurement should be set to approx. 10 min (see Notes 9 and 10). 2. Time-lapse sequences were recorded with FluoView Tiempo (v4.3) time course software at room temperature.
3.3 Calcium Signal Analysis
1. Fix the chamber, and select an appropriate microscopic field (field of view) (see Notes 11 and 12). 2. Start scanning in XYT mode for about 1 min to determine baseline values.
Adrienn Pe´ntek et al.
3. Prepare 500 μM ATP solution: Add 0.5 μL of 100 mM ATP to 100 μL HBSS (see Note 3). 4. Add 50 μL of 500 μM ATP to the first well (final concentration 100 μM) very carefully (see Note 13), drop by drop, and continue scanning until calcium signal returns to baseline level (it takes a few minutes) (see Note 14). 5. The maximum and minimum levels of the calcium-dependent cellular fluorescence can be estimated in the presence of ionomycin and after the addition of excess EGTA to the medium, respectively. Prepare 30 μM ionomycin solution in HBSS: add 3 μL 1 mM ionomycin to 100 μL HBSS. Add 50 μL of this ionomycin solution to the cells very carefully (final concentration 5 μM). 6. After fluorescence intensity reaches its maximum use 33 μL 100 mM EGTA solution (final concentration 10 mM) to get the minimum fluorescence intensity level. 7. When fluorescence intensity reaches its minimum stop recording and save the experiment. 8. Repeat dye loading and calcium measurement in each well (see Note 15). 3.4
4
Data Analysis
Region of interest (ROI) was selected for each hESC clump (see Fig. 1) and was analyzed with the FluoView Tiempo (v4.3, Olympus, http://www.olympusmicro.com) software (see Note 16). Microsoft Excel software was used to analyze raw data. Relative fluorescence was calculated as percentage between maximal and minimal fluorescence: ((F Fmin) 100)/(F (Fmax Fmin)) (where Fmax was the maximal fluorescence after addition of ionomycin and Fmin was the minimal fluorescence after addition of EGTA) (Fig. 1b, c and 2).
Notes 1. Weigh 0.25 g gelatin (type A, from porcine, Sigma) and transfer to the glass beaker. Add bi-distillated water to a volume of 200 mL. Mix, and heat gently. Make up to 250 mL with bi-distillated water after the solution has cleared up. Sterilize the solution in autoclave or by filtering. 2. Solve 50 μg Fluo4-AM (Molecular Probes) in 50 μL DMSO. Store at 20 C. For calcium signal measurements Fluo4-AM was used at a 1 μM final concentration. 3. The ATP stock solution (100 mM) should be stored at 20 C in aliquots. ATP solution was diluted freshly before experiment and stored at 4 C. ATP was used at a 100 μM final concentration.
Analysis of Calcium Signaling
Fig. 1 Calcium signals in Fluo4-AM-loaded HUES 9 human embryonic stem cells. (a) Consecutive images were made after addition of ATP, ionomycin, and EGTA, respectively, as indicated in the pictures. The confocal images were artificially colored for better visualization of changes in calcium level. (b) Analysis of confocal microscopy imaging data was performed in the areas of hESC clumps assigned in (a) panel. (c) Statistical analysis of calcium signals measured on hESC clumps. Values represent the means S.D. of three independent experiments (13 clumps)
Fig. 2 Calcium signals in Fluo4-AM-loaded human induced pluripotent stem cells. Statistical analysis of calcium signals measured on hiPSC clumps. Values represent the means S.D. of three independent experiments (nine clumps)
Adrienn Pe´ntek et al.
4. Weigh 9.50875 g Titriplex VI (Merck) and transfer to the glass beaker. Add bi-distillated water to a volume of 250 mL. Mix, and adjust pH with 1.7 M Tris. 5. We found that culturing hESCs on an MEF layer is the most reliable method for calcium measurements. Cells might partially differentiate on a glass surface and shift more easily during treatments with ionomycin and EGTA without a feeder layer. Feeder cells can be simply excluded from the analysis according to morphological differences (Fig. 1a). 6. To avoid overgrowth of hESC clumps do not seed too many clumps into wells. The overgrown clumps can be partially differentiated on glass surface, and the loading of the fluorescent dye can be inhomogeneous within bigger clumps. 7. Diluted Fluo4-AM solution is not stable, and thus it is worth diluting it freshly each time. 8. Dye loading medium (KO-DMEM) and HBSS solution do not contain serum or serum replacement; thus, the hESC cells might starve during the experiment. Hence keep the cells in serum-deprived medium as short as possible. 9. The calcium signals can be studied by any other microscopic systems (for example wide-field fluorescent microscope); however, we found that the abovementioned confocal microscopic settings reflect calcium signals more accurately than others. Similar result has been found recently studying DT40 lymphocytes using GECI (15). 10. The calcium signals can be performed using GECIs, as well (16), utilizing the same confocal microscopic settings. 11. Fixing the chamber on the microscope is crucial; otherwise, any motion (e.g., when reagents are added) might fail the experiment. 12. The optimal field of view contains medium-size, compact hESC clumps (Fig. 1a). 13. Alternatively, calcium-free HBSS might be used to measure store-operated calcium entry (SOCE) (17). 14. The exact time of recording should be optimized for each calcium signal-inducing agent. Wait until the signal declines and a new equilibrium is reached. 15. We have used this method for studying calcium signals in induced pluripotent cells (18) as well (Fig. 2). 16. Images can be processed with ImageJ software as well.
Analysis of Calcium Signaling
Acknowledgments The authors appreciate the gift of HUES9 cell line by Dr. Douglas Melton, HHMI. This work was supported by the Hungarian Scientific Research Fund [grant numbers NK83533 and CK80283] and by the National Development Agency [grant numbers KTIA_AIK_12-1-2012-0025, KMR_12-1-2012-0112]. References 1. Dolmetsch RE, Xu K, Lewis RS (1998) Calcium oscillations increase the efficiency and specificity of gene expression. Nature 392:933–936 2. Feske S, Giltnane J, Dolmetsch R et al (2001) Gene regulation mediated by calcium signals in T lymphocytes. Nat Immunol 2:316–324 3. Qu B, Al-Ansary D, Kummerow C et al (2011) ORAI-mediated calcium influx in T cell proliferation, apoptosis and tolerance. Cell Calcium 50:261–269 4. Illes P, Alexandre Ribeiro J (2004) Molecular physiology of P2 receptors in the central nervous system. Eur J Pharmacol 483:5–17 5. Grynkiewicz G, Poenie M, Tsien RY (1985) A new generation of Ca2+ indicators with greatly improved fluorescence properties. J Biol Chem 260:3440–3450 6. Takahashi A, Camacho P, Lechleiter JD et al (1999) Measurement of intracellular calcium. Physiol Rev 79:1089–1125 7. Horikawa K, Yamada Y, Matsuda T et al (2010) Spontaneous network activity visualized by ultrasensitive Ca(2+) indicators, yellow Cameleon-Nano. Nat Methods 7:729–732 8. Miyawaki A, Llopis J, Heim R et al (1997) Fluorescent indicators for Ca2+ based on green fluorescent proteins and calmodulin. Nature 388:882–887 9. Nagai T, Yamada S, Tominaga T et al (2004) Expanded dynamic range of fluorescent indicators for Ca(2+) by circularly permuted yellow fluorescent proteins. Proc Natl Acad Sci U S A 101:10554–10559 10. Paredes RM, Etzler JC, Watts LT et al (2008) Chemical calcium indicators. Methods 46:143–151
11. Thomas D, Tovey SC, Collins TJ et al (2000) A comparison of fluorescent Ca2+ indicator properties and their use in measuring elementary and global Ca2+ signals. Cell Calcium 28:213–223 12. Apati A, Paszty K, Erdei Z et al (2011) Calcium signaling in pluripotent stem cells. Mol Cell Endocrinol 353:57–67 13. Malmersjo S, Liste I, Dyachok O et al (2010) Ca2+ and cAMP signaling in human embryonic stem cell-derived dopamine neurons. Stem Cells Dev 19:1355–1364 14. Apati A, Orban TI, Varga N et al (2008) High level functional expression of the ABCG2 multidrug transporter in undifferentiated human embryonic stem cells. Biochim Biophys Acta 1778:2700–2709 15. Miyamoto A, Bannai H, Michikawa T et al (2013) Optimal microscopic systems for longterm imaging of intracellular calcium using a ratiometric genetically-encoded calcium indicator. Biochem Biophys Res Commun 434:252–257 16. Apati A, Paszty K, Hegedus L et al (2013) Characterization of calcium signals in human embryonic stem cells and in their differentiated offspring by a stably integrated calcium indicator protein. Cell Signal 25: 752–759 17. Bird GS, DeHaven WI, Smyth JT et al (2008) Methods for studying store-operated calcium entry. Methods 46:204–212 18. Grabundzija I, Wang J, Sebe A et al (2013) Sleeping Beauty transposon-based system for cellular reprogramming and targeted gene insertion in induced pluripotent stem cells. Nucleic Acids Res 41:1829–1847
Analysis of Intracellular Calcium Signaling in Human Embryonic Stem Cells Adrienn Pe´ntek, Katalin Pa´szty, and A´gota Apa´ti Abstract Measurement of changes in intracellular calcium concentration is one of the most common and useful tools for studying signal transduction pathways or cellular responses in basic research and drug screening purposes as well. Increasing number of such applications using human pluripotent stem cells and their derivatives requires development of calcium signal measurements for this special cell type. Here we describe a modified protocol for analysis of calcium signaling events in human embryonic stem cells, which can be used for other pluripotent cell types (such as iPSC) or their differentiated offspring as well. Keywords: Human embryonic stem cell, Calcium signaling, Fluorescent calcium indicators, Genetically encoded calcium indicators, Confocal microscopy
1
Introduction Calcium signals control diverse cellular processes, for example regulation of gene expression (1, 2), cell division or apoptosis (3), and activation of excitatory cell types (4). Analysis of these versatile patterns of calcium signals is essential for studying the mechanisms lying beyond the cellular functions. There are generally two widely used classes of calcium indicators, chemical calcium indicators (5, 6) and genetically encoded calcium indicators (GECIs) (7–9) for calcium imaging. Both types of indicators exhibit altered fluorescent properties according to changes in cytoplasmic calcium concentration. Selection of proper calcium indicator depends on the amplitude, frequency, and spatial and temporal properties of the Ca2+ transient; however, the “global” Ca2+ signals can be measured by most of the commonly used calcium indicators (10, 11). Human embryonic stem cell lines have normal genetic background and can differentiate toward various cell types which make them a favorable model system for studying calcium signals evoked by endogenous substrates and a wide range of drugs. At the same time there are only limited numbers of publications about calcium signals in human pluripotent stem cells (12, 13). This is partly due to the special feature of these cell types such as the formation of
Adrienn Pe´ntek et al.
three-dimensional clumps, presence of feeder cells, sensitivity of human PSCs to any enzymatic or mechanical cell separation, and heterogeneous shape and function of the PSCs even in a given clump (cell aggregate). Here we present a commonly usable method based on confocal microscopic system which eliminates the abovementioned problems and can be used for calcium signal analysis of hESC and hiPSC lines too.
2
Materials
2.1 Maintenance of hES Cells
All media were sterile filtered with 0.22 μm Steritop-GP (Millipore) vacuum filter units. All media and reagents were added at room temperature. 1. 0.1 % gelatin solution in water (see Note 1). 2. Feeder cell layer: Mouse embryonic fibroblast (MEF) cells isolated from embryos at day 13 (Millipore) and treated by Mitomycin C (Sigma). 3. MEF culturing medium: 90 % Dulbecco’s Modified Eagle Medium (DMEM, Gibco) supplemented with 10 % Fetal Bovine Serum (Gibco), 1 mM Glutamax-I (Gibco). Store at 4 C. The medium may be used within 2 weeks of preparation. 4. hES culturing medium: 80 % Knockout Dulbecco’s Modified Eagle Medium (KoDMEM, Gibco) supplemented with 15 % Knockout Serum Replacement (Gibco), 1 mM Glutamax-I (Gibco), 0.1 mM beta-mercaptoethanol, 1 % nonessential amino acids, and 4 ng/mL human fibroblast growth factor (Invitrogen). Store at 4 C. The medium may be used within 2 weeks of preparation. 5. hES cells were propagated on mitomycin C-treated MEF feeder layer by enzymatic dissociation with 0.05 % trypsin–EDTA (Invitrogen) and replated on fresh feeder layer every second day as described earlier (14). 6. For confocal microscopic measurements cells were seeded onto an eight-well Nunc Lab-Tek II Chambered Coverglass (Nalgene Nunc International).
2.2 Calcium Signal Analysis in hES Cells
1. Fluo4-AM: 1 mM solution in dimethyl sulfoxide (DMSO) (see Note 2). 2. Hank’s balanced salt solution (HBSS) with calcium and magnesium, without phenol red (Invitrogen). Store at room temperature. 3. 100 mM adenosine-50 -triphosphate (ATP) solution (GE Life Sciences) (see Note 3).
Analysis of Calcium Signaling
4. Ionomycin (Invitrogen): 1 mM solution in DMSO (Invitrogen). Store at 20 C. Ionomycin was used at a 5 μM final concentration for measurements. 5. EGTA: 100 mM solution in water, pH 7.4 (see Note 4).
3
Methods
3.1 Loading of hESCs with the Calcium Indicator, Fluo4-AM
1. Cover the eight-well chambered cover glass with 250 μL of 0.1 % gelatin solution for each well. 2. Store the coated chamber at room temperature under laminar flow cabinet for 2 h. 3. Remove the remaining gelatin solution, and seed mitomycin C-treated MEF cells onto the wells (4 104 cell/well in 250 μL MEF culturing medium). Prepare the MEF feeder layer at least 1 day before plating the hESCs (see Note 5). 4. Seed hESC cells (20–40 clumps/well) on to the mitomycin C-treated MEF feeder layer in 500 μL hES culturing medium 24–48 h prior to calcium signal measurement (see Note 6). 5. On the day of experiment dilute 0.3 μL of 1 mM Fluo4-AM in 300 μL FBS-free KoDMEM (1 μM final concentration). Keep this solution at room temperature in the dark (see Note 7). 6. Remove the hES culturing medium, add 200 μL diluted Fluo4AM dye solution to one well, and keep the chamber in a CO2 incubator for 15–30 min. 7. Wash the cells with HBSS before confocal imaging. 8. Finally, add 200 μL of HBSS solution to the cells and place the chamber onto the microscope (see Note 8).
3.2 Microscope Settings
1. All settings are adjusted to an Olympus IX-81 laser scanning confocal microscope using an Olympus PLAPO 20 objective. For Fluo4 imaging excite cells at 488 nm and collect emission data between 505 and 535 nm. Set image resolution to 512 512 or lower and scan speed to fast (about 1 s/scan) so that time resolution might be around 1 s. z-resolution should be 4 μm, and the duration of the measurement should be set to approx. 10 min (see Notes 9 and 10). 2. Time-lapse sequences were recorded with FluoView Tiempo (v4.3) time course software at room temperature.
3.3 Calcium Signal Analysis
1. Fix the chamber, and select an appropriate microscopic field (field of view) (see Notes 11 and 12). 2. Start scanning in XYT mode for about 1 min to determine baseline values.
Adrienn Pe´ntek et al.
3. Prepare 500 μM ATP solution: Add 0.5 μL of 100 mM ATP to 100 μL HBSS (see Note 3). 4. Add 50 μL of 500 μM ATP to the first well (final concentration 100 μM) very carefully (see Note 13), drop by drop, and continue scanning until calcium signal returns to baseline level (it takes a few minutes) (see Note 14). 5. The maximum and minimum levels of the calcium-dependent cellular fluorescence can be estimated in the presence of ionomycin and after the addition of excess EGTA to the medium, respectively. Prepare 30 μM ionomycin solution in HBSS: add 3 μL 1 mM ionomycin to 100 μL HBSS. Add 50 μL of this ionomycin solution to the cells very carefully (final concentration 5 μM). 6. After fluorescence intensity reaches its maximum use 33 μL 100 mM EGTA solution (final concentration 10 mM) to get the minimum fluorescence intensity level. 7. When fluorescence intensity reaches its minimum stop recording and save the experiment. 8. Repeat dye loading and calcium measurement in each well (see Note 15). 3.4
4
Data Analysis
Region of interest (ROI) was selected for each hESC clump (see Fig. 1) and was analyzed with the FluoView Tiempo (v4.3, Olympus, http://www.olympusmicro.com) software (see Note 16). Microsoft Excel software was used to analyze raw data. Relative fluorescence was calculated as percentage between maximal and minimal fluorescence: ((F Fmin) 100)/(F (Fmax Fmin)) (where Fmax was the maximal fluorescence after addition of ionomycin and Fmin was the minimal fluorescence after addition of EGTA) (Fig. 1b, c and 2).
Notes 1. Weigh 0.25 g gelatin (type A, from porcine, Sigma) and transfer to the glass beaker. Add bi-distillated water to a volume of 200 mL. Mix, and heat gently. Make up to 250 mL with bi-distillated water after the solution has cleared up. Sterilize the solution in autoclave or by filtering. 2. Solve 50 μg Fluo4-AM (Molecular Probes) in 50 μL DMSO. Store at 20 C. For calcium signal measurements Fluo4-AM was used at a 1 μM final concentration. 3. The ATP stock solution (100 mM) should be stored at 20 C in aliquots. ATP solution was diluted freshly before experiment and stored at 4 C. ATP was used at a 100 μM final concentration.
Analysis of Calcium Signaling
Fig. 1 Calcium signals in Fluo4-AM-loaded HUES 9 human embryonic stem cells. (a) Consecutive images were made after addition of ATP, ionomycin, and EGTA, respectively, as indicated in the pictures. The confocal images were artificially colored for better visualization of changes in calcium level. (b) Analysis of confocal microscopy imaging data was performed in the areas of hESC clumps assigned in (a) panel. (c) Statistical analysis of calcium signals measured on hESC clumps. Values represent the means S.D. of three independent experiments (13 clumps)
Fig. 2 Calcium signals in Fluo4-AM-loaded human induced pluripotent stem cells. Statistical analysis of calcium signals measured on hiPSC clumps. Values represent the means S.D. of three independent experiments (nine clumps)
Adrienn Pe´ntek et al.
4. Weigh 9.50875 g Titriplex VI (Merck) and transfer to the glass beaker. Add bi-distillated water to a volume of 250 mL. Mix, and adjust pH with 1.7 M Tris. 5. We found that culturing hESCs on an MEF layer is the most reliable method for calcium measurements. Cells might partially differentiate on a glass surface and shift more easily during treatments with ionomycin and EGTA without a feeder layer. Feeder cells can be simply excluded from the analysis according to morphological differences (Fig. 1a). 6. To avoid overgrowth of hESC clumps do not seed too many clumps into wells. The overgrown clumps can be partially differentiated on glass surface, and the loading of the fluorescent dye can be inhomogeneous within bigger clumps. 7. Diluted Fluo4-AM solution is not stable, and thus it is worth diluting it freshly each time. 8. Dye loading medium (KO-DMEM) and HBSS solution do not contain serum or serum replacement; thus, the hESC cells might starve during the experiment. Hence keep the cells in serum-deprived medium as short as possible. 9. The calcium signals can be studied by any other microscopic systems (for example wide-field fluorescent microscope); however, we found that the abovementioned confocal microscopic settings reflect calcium signals more accurately than others. Similar result has been found recently studying DT40 lymphocytes using GECI (15). 10. The calcium signals can be performed using GECIs, as well (16), utilizing the same confocal microscopic settings. 11. Fixing the chamber on the microscope is crucial; otherwise, any motion (e.g., when reagents are added) might fail the experiment. 12. The optimal field of view contains medium-size, compact hESC clumps (Fig. 1a). 13. Alternatively, calcium-free HBSS might be used to measure store-operated calcium entry (SOCE) (17). 14. The exact time of recording should be optimized for each calcium signal-inducing agent. Wait until the signal declines and a new equilibrium is reached. 15. We have used this method for studying calcium signals in induced pluripotent cells (18) as well (Fig. 2). 16. Images can be processed with ImageJ software as well.
Analysis of Calcium Signaling
Acknowledgments The authors appreciate the gift of HUES9 cell line by Dr. Douglas Melton, HHMI. This work was supported by the Hungarian Scientific Research Fund [grant numbers NK83533 and CK80283] and by the National Development Agency [grant numbers KTIA_AIK_12-1-2012-0025, KMR_12-1-2012-0112]. References 1. Dolmetsch RE, Xu K, Lewis RS (1998) Calcium oscillations increase the efficiency and specificity of gene expression. Nature 392:933–936 2. Feske S, Giltnane J, Dolmetsch R et al (2001) Gene regulation mediated by calcium signals in T lymphocytes. Nat Immunol 2:316–324 3. Qu B, Al-Ansary D, Kummerow C et al (2011) ORAI-mediated calcium influx in T cell proliferation, apoptosis and tolerance. Cell Calcium 50:261–269 4. Illes P, Alexandre Ribeiro J (2004) Molecular physiology of P2 receptors in the central nervous system. Eur J Pharmacol 483:5–17 5. Grynkiewicz G, Poenie M, Tsien RY (1985) A new generation of Ca2+ indicators with greatly improved fluorescence properties. J Biol Chem 260:3440–3450 6. Takahashi A, Camacho P, Lechleiter JD et al (1999) Measurement of intracellular calcium. Physiol Rev 79:1089–1125 7. Horikawa K, Yamada Y, Matsuda T et al (2010) Spontaneous network activity visualized by ultrasensitive Ca(2+) indicators, yellow Cameleon-Nano. Nat Methods 7:729–732 8. Miyawaki A, Llopis J, Heim R et al (1997) Fluorescent indicators for Ca2+ based on green fluorescent proteins and calmodulin. Nature 388:882–887 9. Nagai T, Yamada S, Tominaga T et al (2004) Expanded dynamic range of fluorescent indicators for Ca(2+) by circularly permuted yellow fluorescent proteins. Proc Natl Acad Sci U S A 101:10554–10559 10. Paredes RM, Etzler JC, Watts LT et al (2008) Chemical calcium indicators. Methods 46:143–151
11. Thomas D, Tovey SC, Collins TJ et al (2000) A comparison of fluorescent Ca2+ indicator properties and their use in measuring elementary and global Ca2+ signals. Cell Calcium 28:213–223 12. Apati A, Paszty K, Erdei Z et al (2011) Calcium signaling in pluripotent stem cells. Mol Cell Endocrinol 353:57–67 13. Malmersjo S, Liste I, Dyachok O et al (2010) Ca2+ and cAMP signaling in human embryonic stem cell-derived dopamine neurons. Stem Cells Dev 19:1355–1364 14. Apati A, Orban TI, Varga N et al (2008) High level functional expression of the ABCG2 multidrug transporter in undifferentiated human embryonic stem cells. Biochim Biophys Acta 1778:2700–2709 15. Miyamoto A, Bannai H, Michikawa T et al (2013) Optimal microscopic systems for longterm imaging of intracellular calcium using a ratiometric genetically-encoded calcium indicator. Biochem Biophys Res Commun 434:252–257 16. Apati A, Paszty K, Hegedus L et al (2013) Characterization of calcium signals in human embryonic stem cells and in their differentiated offspring by a stably integrated calcium indicator protein. Cell Signal 25: 752–759 17. Bird GS, DeHaven WI, Smyth JT et al (2008) Methods for studying store-operated calcium entry. Methods 46:204–212 18. Grabundzija I, Wang J, Sebe A et al (2013) Sleeping Beauty transposon-based system for cellular reprogramming and targeted gene insertion in induced pluripotent stem cells. Nucleic Acids Res 41:1829–1847
