Original Article
Improved Method to Retain Cytosolic Reporter Protein Fluorescence While Staining for Nuclear Proteins Andre P. Heinen,1 Florian Wanke,1 Sonja Moos,1 Sebastian Attig,2 Herve Luche,3 Prajna Paramita Pal,4 Nediljko Budisa,5 Hans J€ org Fehling,3 Ari Waisman,1 Florian C. Kurschus1*
1
Institute for Molecular Medicine, University Medical Center of the Johannes Gutenberg, University of Mainz, 55131 Mainz, Germany
2
TRON gGmbH—Translational Oncology at the University Medical Center of the Johannes Gutenberg University Mainz, 55131 Mainz, Germany
3
Institute of Immunology, University Clinics Ulm, Ulm, Germany
4
Department of Molecular Biotechnology, Max-Planck Institute of Biochemistry, Martinsried, Germany
5
Department of Chemistry/Biokatalysis, Berlin Institute of Technology/TU Berlin, Berlin, Germany
Received 28 October 2013; Revised 28 January 2014; Accepted 1 February 2014 Grant sponsor: German Research Foundation, Grant numbers: TR 128 TPA3 and TPA7, TR 52 TPC2. Additional Supporting Information may be found in the online version of this article. This work was partially presented as poster during the Annual Meeting of the German Society of Immunology in Mainz in 2013. Present Address for Herv e Luche: Present address of Herve Luche: Centre for Immunophenomic, INSERM US012/CNRS, AMU UMS3367, Marseille, France Grant sponsor: German Research Foundation, Grant numbers: TR 128 TPA3 and TPA7, TR 52 TPC2.
Cytometry Part A 85A: 621627, 2014
Abstract Staining of transcription factors (TFs) together with retention of fluorescent reporter proteins is hindered by loss of fluorescence using current available methods. In this study, it is shown that current TF staining protocols do not destroy fluorescent proteins (FPs) but rather that fixation is not sufficient to retain FPs in the cytosol of the permeabilized cells. In this article, a simple and reliable protocol is elaborated, which allows efficient TF and cytokine staining while retaining FPs inside fixed cells. VC 2014 International Society for Advancement of Cytometry
Key terms EYFP; RFP; EGFP; FoxP3; RORct; T-bet; flow cytometry; fixation; fate mapping; formaldehyde; mouse T cells
TO
address the functions of cells and genes and their expression patterns, reporter mice are used. These mice often express cytosolic-soluble fluorescent proteins (FPs) under direct control of a tissue- or cell-type-specific promoter. Alternatively, and often for fate mapping projects, mice with conditional expression of FPs from the Rosa26 locus are used (1). Because of a loxP-flanked transcriptional STOP cassette preceding the FP gene, the latter is only expressed in cells in which a specific Cre-recombinase is or was active. Fate mapping mice constitute a very useful tool to study cell differentiation and to trace cell destiny/progeny, because of the irreversible tagging once the respective Cre was produced (2). As there is significant interest in staining lineage decision transcription factors (TFs) in fate mapping experiments, we thought to develop a method which would allow simultaneous staining of nuclear TFs such as FoxP3, RORct, or T-bet in cells which express a fluorescent reporter protein. In contrast to the common understanding, we found that the usual methods of TF detection by flow cytometry do not destroy fluorescence of the reporter proteins but rather rely on the use of too weak fixation conditions. This insufficient fixation does not retain FPs inside the cells when they are permeabilized during the TF-staining procedure. This is corroborated by the fact that reporter proteins, which are bound to the cell membrane as part of fusion proteins, for example, in case of the DTRGFP in DEREG mice (3), do not lose their fluorescence on FoxP3 staining using standard FoxP3 staining protocols (3). The inherent problem seems that stronger fixation, commonly used for cytokine staining, which is necessary to keep cytosolic proteins confined within cells during permeabilization, hinders or masks efficient staining of TFs. By varying the fixative with different timings and concentrations, we were able to define an intermediate fixation strength, which
Original Article
*Correspondence to: Florian C. Kurschus, Institute for Molecular Medicine, Obere Zahlbacher Str. 67, 55131 Mainz, Germany. E-mail: [email protected]
DOI: 10.1002/cyto.a.22451 C 2014 International Society for Advancement of Cytometry V
Published online 19 February 2014 in Wiley Online Library (wileyonlinelibrary.com)
retains fluorescent cytosolic reporter proteins inside the cell, allowing simultaneous staining of cytokines and TFs.
MATERIALS AND METHODS Mice and animal experiments The following transgenic mouse strains were used and described previously: CD4-Cre (4), Rosa26-EYFP (1), and Rosa26-RFP (conditional tdRFP) (5). All strains were backcrossed to the C57BL/6 background. WT-C57BL/6 animals were bred in-house. All animal experiments were performed in accordance with our license of the government agency for animal welfare of Rhineland-Palatinate (Mainz, Germany). T-cell purification Single-cell suspension of splenocytes and thymocytes from na€ıve or myelin oligodendrocyte glycoprotein in complete Freund’s adjuvant immunized mice were nonenzymatically prepared in Dulbecco’s Phosphate Buffered Saline with 2% fetal calf serum. Hereafter, red blood cell lysis was performed in 1 3 ACK buffer (150 mM ammonium chloride, 10 mM potassium bicarbonate, and 1 mM Triplex III) for 3 min on ice. CNS-infiltrating cells in mice with experimental autoimmune encephalomyelitis (EAE) were purified similarly as previously described (6). Bead-based capture assay Magnetic protein-G-bound Dynal beads (Dynal, life technologies: Darmstadt, Germany, # 10004D) were diluted to 1:100 in staining buffer (DPBS, 1% BSA and 0.1% NaN3) and bound to polyclonal rabbit anti-GFP (#ab6556; Abcam, Cambridge, United Kingdom) diluted to 1:500 in staining buffer. Thereafter, antibody-bound beads were incubated with either recombinant EGFP [rEGFP; prepared similarly as previously described (Ref. 7))] or with 100 mL of supernatants of buffer-treated cells in a total volume of 200 mL for 2 h at 4 C. Immunization Mice were immunized subcutaneously with 50 mg of MOG35–55 peptide in 100 mL CFA containing 1.1 mg of heatkilled Mycobacterium tuberculosis. For EAE induction, additionally 200 ng of pertussis toxin (Sigma-Aldrich, Taufkirchen, Germany) was injected intraperitoneally on Days 0 and 2 post immunization. Flow cytometry staining and acquisition Antibodies for flow cytometry were purchased from eBioscience (Frankfurt, Germany), BD Pharmingen (Heidelberg, Germany), or BioLegend (Fell, Germany). For intracellular cytokine (IC) staining, cells were activated for 4 h in phorbol-12-myristate-13-acetate (PMA) (50 ng/mL) and Ion622
omycin (500 ng/mL) in the presence of Brefeldin A (1 mg/mL) before surface staining. All incubations were performed on ice or at 4 C and in the dark. Prior to surface staining, all cells were pretreated with FC-block at 5 mg/mL (BioXCell Cologne, Germany clone 2.4G2) for 10 min. For surface staining, the following antibodies were used: CD90.2-APCeFluor780 or Pacific Blue (PB) (clone 53-2.1), CD4-PerCP or PECy5 or V500 (clone GK1.5), CD4-BV510 (clone RM4–5), CD8-PECy7 or V450 or V500 (clone 53-6.7), CD25-APC (clone PC61), CD11b-PE-Cy7 or eFluor450 (clone M1/70), and CD45.2-APCeFluor780 (clone 30-F11), diluted in staining buffer and stained for 15 min. Cells were washed and fixed either in 1% or 2% phosphate buffered paraformaldehyde (PFA; catalog # 0335; Carl Roth, Karlsruhe, Germany) or phosphate buffered formaldehyde (FA; diluted from RotiVHistofix 4%, which contains less than 3% methanol as stabilization reagent; catalog # P087; Carl Roth, Germany). Working concentration of fixative was obtained by diluting the 4% stock solution with DPBS directly prior to use. Cells were permeabilized using a 0.1% saponin buffer (DPBS, 0.1% saponin, 1% BSA, and 0.1% NaN3) or 1 3 eBio-Perm buffer (catalog # 00–8333; eBioscience). Thereafter, cells were stained intracellularly (IC) for FoxP3-FITC or PE or APC or eFluor450 (clone Fjk-16s), T-bet-eFluor660 (clone ebio4B10), RORct-PE or APC or PerCPeFluor710 (clone B2D), IL-17A-PE or eFluor450 (clone ebio17B7), and IFN-c-PE-Cy7 or APC (clone XMG1.2) in 0.1% saponin buffer or 1 3 eBio-Perm for 30 min or O/N. After this incubation step, cells were washed twice, using the same buffer as for intracellular staining. Acquisition of stained cells was performed in staining buffer using a BD FACScan flow cytometer, a BD FACSCanto II flow cytometer, or BD LSRFortessa cytometer. BD FACSCanto II setup: blue laser (488 nm), filters: 488/10, 530/30, 585/42, 670 LP, 780/60; red laser (640 nm), filters: 660/20, 780/60; violet laser (405 nm), filters: 450/50, 510/50. BD LSRFortessa setup: blue laser (488 nm), filters: 488/10, 530/30, 710/50; red laser (640 nm), filters: 670/30, 730/45, 780/60; yellow/green laser (561 nm), filters: 586/15, 610/20, 670/30, 710/50, 780/ 60; and violet laser (405 nm), filters: 450/50, 525/50, 610/20, 670/30, 710/50. Unstained cells and/or compensation beads (catalog # 552843 or # 552845; CompBeads BD) and single fluorochrome-stained cells and/or CompBeads were used to generate the compensation matrix. For fcs-file analysis, FlowJo version 8.8.7 (Tree Star) was used. Our gating strategy was to gate first on desired lymphocytes, then we gated on singlets using the ability of forward scatter (FSC)-H vs. FSCA, and from this gate, we analyzed T cells as indicated in the figure legends. Stain index (Si) was calculated using the following formula: R
Transcription Factor Staining with Retention of Fluorescent Proteins
Original Article
Si 5 ðMFI1 2MFI2 Þ=ð23SD of MFI2 Þ: Statistics Graphs were made with Prism version 5.0b (GraphPhad). Mean values of replicates with standard deviation are shown. Statistical significance was calculated using the unpaired twotailed t-test.
RESULTS To address the issue of loss of fluorescence of reporter proteins when staining for TFs, we first tested different fixation/permeabilization protocols. Using splenocytes from CD4-Cre 3 EYFPFl/1 (CD4/EYFP) mice (Fig. 1a), we found that fluorescence of EYFP was strongest when cells were analyzed in a native state without fixation or permeabilization. Fluorescence was completely lost by usage of the FoxP3 staining kit from eBioscience, which uses two steps. A first one in which cells are simultaneously fixed and permeabilized (eBioFix/Perm), and a second step in which cells are only kept permeabilized for TF staining (eBio-Perm). We noticed that fluorescence is largely maintained by the use of 2% of PFAderived FA (subsequently referred as PFA) as fixative, irrespective of the subsequent fixation or permeabilization (Fig. 1a). Importantly, a prefixation step using 2% PFA before the eBioFix/Perm and Perm steps also rescued EYFP fluorescence (Fig. 1b). This indicated that loss of fluorescence associated with the use of eBio-Fix/Perm buffer is not due to destruction of the fluorophore but rather due to insufficient retention of the FP inside the cell. To analyze this in more detail, we tested the influence of the various fixation/permeabilization buffers on rEGFP and on the release of proteins by EYFP-expressing T cells (Supporting Information Fig. S1). Fluorescence of rEGFP was only partially lost (25%) after incubation in eBio-Fix/ Perm (Supporting Information Fig. S1a), which cannot explain the complete loss of EYFP in cells treated with the FoxP3 kit. Analysis of the concentration of proteins washed out by eBio-Fix/Perm showed a higher protein concentration in supernatants of cells treated in eBio-Fix/Perm than in supernatants of cells treated with 0.5% Triton X-100 (Supporting Information Fig. S1b). This indicates a major leakage of protein from permeabilized cells, which were insufficiently fixed in this buffer. We further established a method to catch rEGFP using Protein G beads bound to anti-GFP antibody (Supporting Information Fig. S1c). Using this assay, we analyzed supernatants of EYFP-positive T cells treated with various buffers and were able to detect EYFP in the supernatant of cells treated with eBio-Perm. In contrast, when such cells were first treated with eBio-Fix/Perm and subsequently with eBioPerm, we failed to detect EYFP in the supernatant (Supporting Information Fig. S1d). Together with the near complete loss of fluorescence of cells treated with eBio-Fix/Perm only (Supporting Information Fig. S1e), this strongly indicates that EYFP is passively washed out in the eBio-Fix/Perm step due to an insufficient fixation in the mixed eBio-Fix/Perm buffer. Although EYFP fluorescence was maintained by fixation in PFA, staining of FoxP3 was reduced in intensity and in Cytometry Part A 85A: 621627, 2014
percentage (Figs. 1a and 1b). Staining of T-bet in CD8 T cells (Fig. 1c) was also dramatically reduced. Double-positive (DP) thymocytes were previously demonstrated to homogeneously express RORct (8). Staining of RORct in DP thymocytes (Fig. 1d) was strongly impaired by fixation in PFA, whereas in CNS-infiltrating CD41 T cells, isolated from mice with EAE, cytokine staining of IFN-c and IL-17A was similar in all intracellular staining methods we tested (Fig. 1e). We then reduced the PFA concentration for fixation and varied fixation time with subsequent permeabilization in the buffer provided by the eBioscience kit (eBio-Perm). Although we found a clear rise in fluorescence by increasing the fixation time (Figs. 2a and 2b), recovery of EYFP was weak and combined with a considerable loss of FoxP3 staining (Figs. 2c and 2d). As formaldehyde solutions, which are derived from fluid formaldehyde stabilized by methanol (subsequently referred as FA), are known to show a differential fixation pattern when compared with solubilized PFA, we decided to test FA as alternative fixative. Therefore, we used FA in different concentrations (data not shown) and fixation times. Indeed, we found that raising fixation time up to 1 h in 2% FA allowed a perfect discrimination of an EYFP-positive population (Figs. 3a and 3b) together with staining for FoxP3 (Fig. 3a), T-bet (Fig. 3b), and RORct (Fig. 3c). Using a not yet described EGFP gene expression reporter mouse strain of our laboratory, we also found that the protocol described here allows simultaneous TF staining with EGFP retention (data not shown). Additionally, we found that the new protocol retained better cellular structure as determined by FSC and side scatter (SSC) in flow cytometry (Supporting Information Fig. S2a), and in addition, surface staining of CD4 and CD8 was also better preserved under conditions of long-time intracellular TF staining when thymocytes were fixed using the protocol reported here (Supporting Information Fig. S2b). As a result, we consistently found a higher yield of cells after the staining procedure when compared with the commercial eBio kit. To test whether the new fixation protocol combined with the eBio-Perm buffer would allow for costaining in the same cells under inflammatory conditions in peripheral T cells, we immunized CD4/EYFP mice with MOG-CFA and recovered dLN cells 9 days later. Indeed, fixation with 2% FA combined with the eBio-Perm buffer retained the same frequency of EYFP-positive cells and allowed costaining of FoxP3, RORct, and T-bet in dLN T cells (Figs. 4a–4d). As expected, Tregs were largely CD25 positive, IL-17Apositive CD41 Th17 cells were mostly RORct positive, whereas IFN-c-positive CD81 T cells coexpressed in majority T-bet (Figs. 4e–4g). We now aimed to test with our new protocol whether tdRFP fluorescent reporter would also be retained when analyzing CNS-infiltrating inflammatory T cells. Therefore, we used conditional RFP reporter mice as previously described (5) and crossed to CD4-Cre (CD4/RFP) mice with EAE (described in “Materials and Methods” section). We sacrificed the mice on Day 11 after disease induction and stained CNSinfiltrating T cells. As shown in Figure 4h, 91% of native 623
Original Article
Figure 1. Defining the problem. Different fixation/permeabilization combinations are shown. (a) CD90.21(APCeFluor780) CD41(PerCP) CD82(V500) splenocytes of a MOG immunized CD4-Cre 3 EYFPFL/1 (CD4/EYFP) mouse. Upper row: EYFP expression [shaded histograms gated on CD90.22(APCeFluor780) cells demonstrate background staining]. Lower row: Percentages of FoxP3(eFluor450)-positive T cells under different conditions are demonstrated. (b) CD41(PECy5) splenic T cells of an EYFP germline deleter mouse are shown. Upper panel: EYFP expression (shaded histogram eBio-Fix/Perm-treated cells shows background). Lower panel: The percentage of FoxP3(PE)1 T cells using double fixation. (c) Percentages of T-bet(eFluor660)-positive splenocytes, gated on CD90.21(APCeFluor780) CD42(PerCP) CD81(V500) T cells, of a MOG-immunized CD4/EYFP mouse. (d) Percentages of RORct(PE) in CD90.21(PB) CD41(PerCP)/CD81(V500) DP thymocytes of a WT mouse are shown (shaded histograms gated on CD90.21(PB) CD41(PerCP) CD82(V500) SP thymocytes show background staining). (e) Cytokine staining for IL-17A(PE) and IFN-c(APC) in CNS-infiltrating CD45.2high (APCeFluor780) CD11b2(eFluor 450) CD41(PerCP) CD82(V500) T cells after EAE induction in a WT mouse at Day 15 (Score 4) is shown. Data are representative of three independent experiments.
624
Transcription Factor Staining with Retention of Fluorescent Proteins
Figure 2. Fixation in 1% PFA partially rescues cytosolic fluorescent proteins but inhibits efficient transcription factor staining. All samples were stained IC using eBio-Perm buffer. (a) CD41(PerCP) CD82(V500) splenocytes of a CD4/EYFP mouse are shown. Upper row: EYFP fluorescence after eBio-Fix/Perm fixation. Lower row: EYFP fluorescence after 1% PFA fixation for different fixation times (shaded histograms gated on CD42(PerCP) CD82(V500) cells show background). (b) Statistic of stain index (Si) for EYFP fluorescence as shown in (a). (c) CD90.21(PB) CD41(PerCP) CD82(V500) splenocytes of a WT mouse are shown. Upper row: CD4(PerCP) vs. FoxP3(APC) after eBio-Fix/Perm fixation. Lower row: CD4(PerCP) vs. FoxP3(APC)-positive cells after fixation in 1% PFA for different fixation times. (d) Graphs of percentages as shown in (c). Data are representative of two independent experiments.
Figure 3. Fixation using 2% FA retains cytosolic fluorescent proteins in the cells and allows efficient transcription factor staining. Stainings of either native, eBio-Fix/Perm fixed cells (60 min) or of cells after fixation for different times with 2% FA are shown. (a) FoxP3(eFluor 450) vs. EYFP in CD90.21(APCeFluor780) CD41(PerCP)CD82(V500) splenocytes of a CD4/EYFP mouse. For comparison, the first plot on the left shows forward scatter area (FSC-A) vs. EYFP on native cells. (b) T-bet(eFluor660) vs. EYFP in CD90.21(APCeFluor780) CD42(PerCP) CD81(V500) splenocytes of CD4/EYFP. (c) RORct(APC) expression in CD90.21(PB) CD41(PerCP)/CD81(V500) DP thymocytes of a WT mouse [shaded histograms gated on CD90.21(PB) CD41(PerCP) CD82(V500) SP thymocytes cells show background staining]. Data are representative of two independent experiments.
Original Article
Figure 4. Staining of in vivo generated inflammatory T-cell populations. (a–c, e and f) Gated CD90.21(APCeFluor780) CD41(V500) CD82(PE-Cy7) T cells, (d and g) CD90.21(APCeFluor780) CD42(V500) CD81(V450) T cells of draining LNs from MOG-CFA immunized CD4/ EYFP mice. (a) Histogram showing EYFP fluorescence in native CD41 T cells [shaded histogram gated on CD90.22(APCeFluor780) CD41(PerCP) CD82(V500) cells demonstrates background staining]. (b and c, e and f) CD41 T cells and (d and g) CD81 T cells are stained using the protocol as shown in Figure 5. Different combinations of cytokines vs. TF and EYFP are presented as indicated. (h–l) Gated CD45.2high(APCeFluor780) CD11b2(PE-Cy7) CNS-infiltrating T cells from MOG-CFA-immunized CD4/RFP mice with EAE. (h) Histogram showing RFP fluorescence in native CD41 T cells [shaded histogram gated on CD45.22(APCeFluor780) CD11b2(PE-Cy7) cells demonstrates background staining]. (i–l) Cells stained using the protocol as shown in Figure 5. (i) Histogram shows a certain loss of RFP fluorescent intensity when compared with native cells but the same percentage of tdRFP-positive CD41 T cells [shaded histogram gated on CD45.2low (APCeFluor780) CD11b2(PE-Cy7) cells demonstrates background staining]. (j) Gating of CD41(BV510) tdRFP1 T cells for presentation of IL17A(eFluor450) vs. RORct(PerCPeFluor710) as shown in (k). (l) Quadrants QI–III shown in dot-plot in (k) were used for further gating.
CD41 T cells (neither fixed nor permeabilized) were RFP positive, and fixation with 2% FA completely rescued FPs (Fig. 4i). Using the advantage of our protocol, we could now gate on CD41 RFP1 cells (Fig. 4j) and analyze these cells for cytokine and TF expression (Figs. 4k and 4l). Interestingly, we found during this early stage of EAE development that T-bet expression correlated with expression of RORct and IL-17A, with the highest expression of T-bet found in RORct1/IL626
17A1 CNS-infiltrating T cells (Fig. 4l). This phenomenon may explain the plasticity of Th17 cells in the CNS (2).
DISCUSSION We herein describe a new and simple method allowing staining of TFs and cytokines simultaneously while retaining fluorescence of reporter proteins. Our method combines Transcription Factor Staining with Retention of Fluorescent Proteins
Original Article
Figure 5. Bench-top protocol for transcription factor and cytokine staining with fluorescent reporter protein retention.
fixation in 2% FA for 40–60 min followed by a permeabilization step and staining/washing steps in commercial eBioPerm buffer (Fig. 5). Using this method, FPs are fixed and retained in the cytosol of the cell. Thereby, fluorescent reporter proteins are not washed out completely during permeabilization/staining in subsequently used eBio-Perm buffer during the following TF staining procedure and they can readily be detected by flow cytometry. By defining a proper fixative with the proper timing for fixation, we succeeded in finding a compromise allowing TF staining under fixation conditions strong enough to maintain cytosolic FPs inside of cells. A previous report similarly presented a method for staining TFs with maintenance of reporter proteins (9). This group defined low PFA concentrations (1–2%) as prefixation step before using the commercial eBio-Fix/Perm and eBio-Perm buffers for TF staining. We also tested 1% PFA for varying times as fixative before permeabilization (without using Fix/Perm from eBioscience) and failed to find a condition with effective retention of fluorescence and reliable FoxP3 staining. Additionally, omitting the Fix/Perm step turns our protocol to be easier, and we believe that usage of FA instead of PFA allows a more subtle compromise in fixation. Furthermore, we additionally demonstrate retention of RFP with simultaneous staining of the TF RORct, which proved to be more difficult to stain than FoxP3. Nevertheless, the conclusions of both reports are similar in that a compromise needs to be defined between a strong enough fixation for FP retention which is weak enough not to hamper nuclear protein detection by immunostaining. Despite keeping the percentage of fluorescent reporter positive cells stable, any kind of fixation conditions used in this study showed a certain loss of fluorescence intensity. This
Cytometry Part A 85A: 621627, 2014
is probably at least partially due to fixation of the FP, which might influence the structure or function of the chromophore as described previously (10) and as shown here. Additionally, a further loss of fluorescence protein might occur during the staining steps in permeabilizing buffer even under optimal prefixation conditions (data not shown). Nevertheless, the signal strength surviving the fixation/permeabilization process is in most cases strong enough for its reliable detection by flow cytometry. Leakage from suboptimally fixed cells constitutes the stronger obstacle for detection of FPs in reporter positive cells costained for nuclear proteins. EYFP has a molecular weight which nearly doubles that of the cytokine IFN-c. Therefore, one might speculate why the bigger protein is easier washed out than the smaller cytokine. Although we have no proof for this, our explanation for this differential leakage is that cytokines are not stored in the cytosol where EYFP is located but that they are located in high concentrations in secretory vesicles and on their way to these vesicles in the ER and the Golgi. This additional membrane barrier, together with the densely packed composition of the vesicle lumen, may also retain cytokines under low fixation conditions inside cells. In summary, we provide a new protocol which enables easy and reliable staining of nuclear TFs retaining soluble cytosolic reporter proteins with fluorescence activity.
ACKNOWLEDGMENTS The authors thank Adriana L€angle from the Technical University Darmstadt for technical assistance and Nives H€ ormann from the Center for Thrombosis and Hemostasis (CTH) Mainz for acquisition with the fluorescent reader.
LITERATURE CITED 1. Srinivas S, Watanabe T, Lin CS, William CM, Tanabe Y, Jessell TM, Costantini F. Cre reporter strains produced by targeted insertion of EYFP and ECFP into the ROSA26 locus. BMC Dev Biol 2001;1:4. 2. Kurschus FC, Croxford AL, Heinen AP, W€ ortge S, Ielo D, Waisman A. Genetic proof for the transient nature of the Th17 phenotype. Eur J Immunol 2010;40:3336–3346. 3. Lahl K, Loddenkemper C, Drouin C, Freyer J, Arnason J, Eberl G, Hamann A, Wagner H, Huehn J, Sparwasser T. Selective depletion of Foxp31 regulatory T cells induces a scurfy-like disease. J Exp Med 2007;204:57–63. 4. Lee PP, Fitzpatrick DR, Beard C, Jessup HK, Lehar S, Makar KW, Perez-Melgosa M, Sweetser MT, Schlissel MS, Nguyen S, et al. A critical role for Dnmt1 and DNA methylation in T cell development, function, and survival. Immunity 2001;15:763– 774. 5. Luche H, Weber O, Nageswara Rao T, Blum C, Fehling HJ. Faithful activation of an extra-bright red fluorescent protein in “knock-in” Cre-reporter mice ideally suited for lineage tracing studies. Eur J Immunol 2007;37:43–53. 6. Cardona AE, Huang D, Sasse ME, Ransohoff RM. Isolation of murine microglial cells for RNA analysis or flow cytometry. Nat Protoc 2006;1:1947–1951. 7. Kurschus FC, Pal PP, B€aumler P, Jenne DE, Wiltschi B, Budisa N. Gold fluorescent annexin A5 as a novel apoptosis detection tool. Cytometry Part A 2009;75A:626– 633. 8. Eberl G, Marmon S, Sunshine MJ, Rennert PD, Choi Y, Littman DR. An essential function for the nuclear receptor RORc(t) in the generation of fetal lymphoid tissue inducer cells. Nat Immunol 2004;5:64–73. 9. Grupillo M, Lakomy R, Geng X, Styche A, Rudert WA, Trucco M, Fan Y. An improved intracellular staining protocol for efficient detection of nuclear proteins in YFP-expressing cells. Biotechniques 2011;51:417–420. 10. Ganguly S, Clayton AH, Chattopadhyay A. Fixation alters fluorescence lifetime and anisotropy of cells expressing EYFP-tagged serotonin1A receptor. Biochem Biophys Res Commun 2011;405:234–237.
627
Improved Method to Retain Cytosolic Reporter Protein Fluorescence While Staining for Nuclear Proteins Andre P. Heinen,1 Florian Wanke,1 Sonja Moos,1 Sebastian Attig,2 Herve Luche,3 Prajna Paramita Pal,4 Nediljko Budisa,5 Hans J€ org Fehling,3 Ari Waisman,1 Florian C. Kurschus1*
1
Institute for Molecular Medicine, University Medical Center of the Johannes Gutenberg, University of Mainz, 55131 Mainz, Germany
2
TRON gGmbH—Translational Oncology at the University Medical Center of the Johannes Gutenberg University Mainz, 55131 Mainz, Germany
3
Institute of Immunology, University Clinics Ulm, Ulm, Germany
4
Department of Molecular Biotechnology, Max-Planck Institute of Biochemistry, Martinsried, Germany
5
Department of Chemistry/Biokatalysis, Berlin Institute of Technology/TU Berlin, Berlin, Germany
Received 28 October 2013; Revised 28 January 2014; Accepted 1 February 2014 Grant sponsor: German Research Foundation, Grant numbers: TR 128 TPA3 and TPA7, TR 52 TPC2. Additional Supporting Information may be found in the online version of this article. This work was partially presented as poster during the Annual Meeting of the German Society of Immunology in Mainz in 2013. Present Address for Herv e Luche: Present address of Herve Luche: Centre for Immunophenomic, INSERM US012/CNRS, AMU UMS3367, Marseille, France Grant sponsor: German Research Foundation, Grant numbers: TR 128 TPA3 and TPA7, TR 52 TPC2.
Cytometry Part A 85A: 621627, 2014
Abstract Staining of transcription factors (TFs) together with retention of fluorescent reporter proteins is hindered by loss of fluorescence using current available methods. In this study, it is shown that current TF staining protocols do not destroy fluorescent proteins (FPs) but rather that fixation is not sufficient to retain FPs in the cytosol of the permeabilized cells. In this article, a simple and reliable protocol is elaborated, which allows efficient TF and cytokine staining while retaining FPs inside fixed cells. VC 2014 International Society for Advancement of Cytometry
Key terms EYFP; RFP; EGFP; FoxP3; RORct; T-bet; flow cytometry; fixation; fate mapping; formaldehyde; mouse T cells
TO
address the functions of cells and genes and their expression patterns, reporter mice are used. These mice often express cytosolic-soluble fluorescent proteins (FPs) under direct control of a tissue- or cell-type-specific promoter. Alternatively, and often for fate mapping projects, mice with conditional expression of FPs from the Rosa26 locus are used (1). Because of a loxP-flanked transcriptional STOP cassette preceding the FP gene, the latter is only expressed in cells in which a specific Cre-recombinase is or was active. Fate mapping mice constitute a very useful tool to study cell differentiation and to trace cell destiny/progeny, because of the irreversible tagging once the respective Cre was produced (2). As there is significant interest in staining lineage decision transcription factors (TFs) in fate mapping experiments, we thought to develop a method which would allow simultaneous staining of nuclear TFs such as FoxP3, RORct, or T-bet in cells which express a fluorescent reporter protein. In contrast to the common understanding, we found that the usual methods of TF detection by flow cytometry do not destroy fluorescence of the reporter proteins but rather rely on the use of too weak fixation conditions. This insufficient fixation does not retain FPs inside the cells when they are permeabilized during the TF-staining procedure. This is corroborated by the fact that reporter proteins, which are bound to the cell membrane as part of fusion proteins, for example, in case of the DTRGFP in DEREG mice (3), do not lose their fluorescence on FoxP3 staining using standard FoxP3 staining protocols (3). The inherent problem seems that stronger fixation, commonly used for cytokine staining, which is necessary to keep cytosolic proteins confined within cells during permeabilization, hinders or masks efficient staining of TFs. By varying the fixative with different timings and concentrations, we were able to define an intermediate fixation strength, which
Original Article
*Correspondence to: Florian C. Kurschus, Institute for Molecular Medicine, Obere Zahlbacher Str. 67, 55131 Mainz, Germany. E-mail: [email protected]
DOI: 10.1002/cyto.a.22451 C 2014 International Society for Advancement of Cytometry V
Published online 19 February 2014 in Wiley Online Library (wileyonlinelibrary.com)
retains fluorescent cytosolic reporter proteins inside the cell, allowing simultaneous staining of cytokines and TFs.
MATERIALS AND METHODS Mice and animal experiments The following transgenic mouse strains were used and described previously: CD4-Cre (4), Rosa26-EYFP (1), and Rosa26-RFP (conditional tdRFP) (5). All strains were backcrossed to the C57BL/6 background. WT-C57BL/6 animals were bred in-house. All animal experiments were performed in accordance with our license of the government agency for animal welfare of Rhineland-Palatinate (Mainz, Germany). T-cell purification Single-cell suspension of splenocytes and thymocytes from na€ıve or myelin oligodendrocyte glycoprotein in complete Freund’s adjuvant immunized mice were nonenzymatically prepared in Dulbecco’s Phosphate Buffered Saline with 2% fetal calf serum. Hereafter, red blood cell lysis was performed in 1 3 ACK buffer (150 mM ammonium chloride, 10 mM potassium bicarbonate, and 1 mM Triplex III) for 3 min on ice. CNS-infiltrating cells in mice with experimental autoimmune encephalomyelitis (EAE) were purified similarly as previously described (6). Bead-based capture assay Magnetic protein-G-bound Dynal beads (Dynal, life technologies: Darmstadt, Germany, # 10004D) were diluted to 1:100 in staining buffer (DPBS, 1% BSA and 0.1% NaN3) and bound to polyclonal rabbit anti-GFP (#ab6556; Abcam, Cambridge, United Kingdom) diluted to 1:500 in staining buffer. Thereafter, antibody-bound beads were incubated with either recombinant EGFP [rEGFP; prepared similarly as previously described (Ref. 7))] or with 100 mL of supernatants of buffer-treated cells in a total volume of 200 mL for 2 h at 4 C. Immunization Mice were immunized subcutaneously with 50 mg of MOG35–55 peptide in 100 mL CFA containing 1.1 mg of heatkilled Mycobacterium tuberculosis. For EAE induction, additionally 200 ng of pertussis toxin (Sigma-Aldrich, Taufkirchen, Germany) was injected intraperitoneally on Days 0 and 2 post immunization. Flow cytometry staining and acquisition Antibodies for flow cytometry were purchased from eBioscience (Frankfurt, Germany), BD Pharmingen (Heidelberg, Germany), or BioLegend (Fell, Germany). For intracellular cytokine (IC) staining, cells were activated for 4 h in phorbol-12-myristate-13-acetate (PMA) (50 ng/mL) and Ion622
omycin (500 ng/mL) in the presence of Brefeldin A (1 mg/mL) before surface staining. All incubations were performed on ice or at 4 C and in the dark. Prior to surface staining, all cells were pretreated with FC-block at 5 mg/mL (BioXCell Cologne, Germany clone 2.4G2) for 10 min. For surface staining, the following antibodies were used: CD90.2-APCeFluor780 or Pacific Blue (PB) (clone 53-2.1), CD4-PerCP or PECy5 or V500 (clone GK1.5), CD4-BV510 (clone RM4–5), CD8-PECy7 or V450 or V500 (clone 53-6.7), CD25-APC (clone PC61), CD11b-PE-Cy7 or eFluor450 (clone M1/70), and CD45.2-APCeFluor780 (clone 30-F11), diluted in staining buffer and stained for 15 min. Cells were washed and fixed either in 1% or 2% phosphate buffered paraformaldehyde (PFA; catalog # 0335; Carl Roth, Karlsruhe, Germany) or phosphate buffered formaldehyde (FA; diluted from RotiVHistofix 4%, which contains less than 3% methanol as stabilization reagent; catalog # P087; Carl Roth, Germany). Working concentration of fixative was obtained by diluting the 4% stock solution with DPBS directly prior to use. Cells were permeabilized using a 0.1% saponin buffer (DPBS, 0.1% saponin, 1% BSA, and 0.1% NaN3) or 1 3 eBio-Perm buffer (catalog # 00–8333; eBioscience). Thereafter, cells were stained intracellularly (IC) for FoxP3-FITC or PE or APC or eFluor450 (clone Fjk-16s), T-bet-eFluor660 (clone ebio4B10), RORct-PE or APC or PerCPeFluor710 (clone B2D), IL-17A-PE or eFluor450 (clone ebio17B7), and IFN-c-PE-Cy7 or APC (clone XMG1.2) in 0.1% saponin buffer or 1 3 eBio-Perm for 30 min or O/N. After this incubation step, cells were washed twice, using the same buffer as for intracellular staining. Acquisition of stained cells was performed in staining buffer using a BD FACScan flow cytometer, a BD FACSCanto II flow cytometer, or BD LSRFortessa cytometer. BD FACSCanto II setup: blue laser (488 nm), filters: 488/10, 530/30, 585/42, 670 LP, 780/60; red laser (640 nm), filters: 660/20, 780/60; violet laser (405 nm), filters: 450/50, 510/50. BD LSRFortessa setup: blue laser (488 nm), filters: 488/10, 530/30, 710/50; red laser (640 nm), filters: 670/30, 730/45, 780/60; yellow/green laser (561 nm), filters: 586/15, 610/20, 670/30, 710/50, 780/ 60; and violet laser (405 nm), filters: 450/50, 525/50, 610/20, 670/30, 710/50. Unstained cells and/or compensation beads (catalog # 552843 or # 552845; CompBeads BD) and single fluorochrome-stained cells and/or CompBeads were used to generate the compensation matrix. For fcs-file analysis, FlowJo version 8.8.7 (Tree Star) was used. Our gating strategy was to gate first on desired lymphocytes, then we gated on singlets using the ability of forward scatter (FSC)-H vs. FSCA, and from this gate, we analyzed T cells as indicated in the figure legends. Stain index (Si) was calculated using the following formula: R
Transcription Factor Staining with Retention of Fluorescent Proteins
Original Article
Si 5 ðMFI1 2MFI2 Þ=ð23SD of MFI2 Þ: Statistics Graphs were made with Prism version 5.0b (GraphPhad). Mean values of replicates with standard deviation are shown. Statistical significance was calculated using the unpaired twotailed t-test.
RESULTS To address the issue of loss of fluorescence of reporter proteins when staining for TFs, we first tested different fixation/permeabilization protocols. Using splenocytes from CD4-Cre 3 EYFPFl/1 (CD4/EYFP) mice (Fig. 1a), we found that fluorescence of EYFP was strongest when cells were analyzed in a native state without fixation or permeabilization. Fluorescence was completely lost by usage of the FoxP3 staining kit from eBioscience, which uses two steps. A first one in which cells are simultaneously fixed and permeabilized (eBioFix/Perm), and a second step in which cells are only kept permeabilized for TF staining (eBio-Perm). We noticed that fluorescence is largely maintained by the use of 2% of PFAderived FA (subsequently referred as PFA) as fixative, irrespective of the subsequent fixation or permeabilization (Fig. 1a). Importantly, a prefixation step using 2% PFA before the eBioFix/Perm and Perm steps also rescued EYFP fluorescence (Fig. 1b). This indicated that loss of fluorescence associated with the use of eBio-Fix/Perm buffer is not due to destruction of the fluorophore but rather due to insufficient retention of the FP inside the cell. To analyze this in more detail, we tested the influence of the various fixation/permeabilization buffers on rEGFP and on the release of proteins by EYFP-expressing T cells (Supporting Information Fig. S1). Fluorescence of rEGFP was only partially lost (25%) after incubation in eBio-Fix/ Perm (Supporting Information Fig. S1a), which cannot explain the complete loss of EYFP in cells treated with the FoxP3 kit. Analysis of the concentration of proteins washed out by eBio-Fix/Perm showed a higher protein concentration in supernatants of cells treated in eBio-Fix/Perm than in supernatants of cells treated with 0.5% Triton X-100 (Supporting Information Fig. S1b). This indicates a major leakage of protein from permeabilized cells, which were insufficiently fixed in this buffer. We further established a method to catch rEGFP using Protein G beads bound to anti-GFP antibody (Supporting Information Fig. S1c). Using this assay, we analyzed supernatants of EYFP-positive T cells treated with various buffers and were able to detect EYFP in the supernatant of cells treated with eBio-Perm. In contrast, when such cells were first treated with eBio-Fix/Perm and subsequently with eBioPerm, we failed to detect EYFP in the supernatant (Supporting Information Fig. S1d). Together with the near complete loss of fluorescence of cells treated with eBio-Fix/Perm only (Supporting Information Fig. S1e), this strongly indicates that EYFP is passively washed out in the eBio-Fix/Perm step due to an insufficient fixation in the mixed eBio-Fix/Perm buffer. Although EYFP fluorescence was maintained by fixation in PFA, staining of FoxP3 was reduced in intensity and in Cytometry Part A 85A: 621627, 2014
percentage (Figs. 1a and 1b). Staining of T-bet in CD8 T cells (Fig. 1c) was also dramatically reduced. Double-positive (DP) thymocytes were previously demonstrated to homogeneously express RORct (8). Staining of RORct in DP thymocytes (Fig. 1d) was strongly impaired by fixation in PFA, whereas in CNS-infiltrating CD41 T cells, isolated from mice with EAE, cytokine staining of IFN-c and IL-17A was similar in all intracellular staining methods we tested (Fig. 1e). We then reduced the PFA concentration for fixation and varied fixation time with subsequent permeabilization in the buffer provided by the eBioscience kit (eBio-Perm). Although we found a clear rise in fluorescence by increasing the fixation time (Figs. 2a and 2b), recovery of EYFP was weak and combined with a considerable loss of FoxP3 staining (Figs. 2c and 2d). As formaldehyde solutions, which are derived from fluid formaldehyde stabilized by methanol (subsequently referred as FA), are known to show a differential fixation pattern when compared with solubilized PFA, we decided to test FA as alternative fixative. Therefore, we used FA in different concentrations (data not shown) and fixation times. Indeed, we found that raising fixation time up to 1 h in 2% FA allowed a perfect discrimination of an EYFP-positive population (Figs. 3a and 3b) together with staining for FoxP3 (Fig. 3a), T-bet (Fig. 3b), and RORct (Fig. 3c). Using a not yet described EGFP gene expression reporter mouse strain of our laboratory, we also found that the protocol described here allows simultaneous TF staining with EGFP retention (data not shown). Additionally, we found that the new protocol retained better cellular structure as determined by FSC and side scatter (SSC) in flow cytometry (Supporting Information Fig. S2a), and in addition, surface staining of CD4 and CD8 was also better preserved under conditions of long-time intracellular TF staining when thymocytes were fixed using the protocol reported here (Supporting Information Fig. S2b). As a result, we consistently found a higher yield of cells after the staining procedure when compared with the commercial eBio kit. To test whether the new fixation protocol combined with the eBio-Perm buffer would allow for costaining in the same cells under inflammatory conditions in peripheral T cells, we immunized CD4/EYFP mice with MOG-CFA and recovered dLN cells 9 days later. Indeed, fixation with 2% FA combined with the eBio-Perm buffer retained the same frequency of EYFP-positive cells and allowed costaining of FoxP3, RORct, and T-bet in dLN T cells (Figs. 4a–4d). As expected, Tregs were largely CD25 positive, IL-17Apositive CD41 Th17 cells were mostly RORct positive, whereas IFN-c-positive CD81 T cells coexpressed in majority T-bet (Figs. 4e–4g). We now aimed to test with our new protocol whether tdRFP fluorescent reporter would also be retained when analyzing CNS-infiltrating inflammatory T cells. Therefore, we used conditional RFP reporter mice as previously described (5) and crossed to CD4-Cre (CD4/RFP) mice with EAE (described in “Materials and Methods” section). We sacrificed the mice on Day 11 after disease induction and stained CNSinfiltrating T cells. As shown in Figure 4h, 91% of native 623
Original Article
Figure 1. Defining the problem. Different fixation/permeabilization combinations are shown. (a) CD90.21(APCeFluor780) CD41(PerCP) CD82(V500) splenocytes of a MOG immunized CD4-Cre 3 EYFPFL/1 (CD4/EYFP) mouse. Upper row: EYFP expression [shaded histograms gated on CD90.22(APCeFluor780) cells demonstrate background staining]. Lower row: Percentages of FoxP3(eFluor450)-positive T cells under different conditions are demonstrated. (b) CD41(PECy5) splenic T cells of an EYFP germline deleter mouse are shown. Upper panel: EYFP expression (shaded histogram eBio-Fix/Perm-treated cells shows background). Lower panel: The percentage of FoxP3(PE)1 T cells using double fixation. (c) Percentages of T-bet(eFluor660)-positive splenocytes, gated on CD90.21(APCeFluor780) CD42(PerCP) CD81(V500) T cells, of a MOG-immunized CD4/EYFP mouse. (d) Percentages of RORct(PE) in CD90.21(PB) CD41(PerCP)/CD81(V500) DP thymocytes of a WT mouse are shown (shaded histograms gated on CD90.21(PB) CD41(PerCP) CD82(V500) SP thymocytes show background staining). (e) Cytokine staining for IL-17A(PE) and IFN-c(APC) in CNS-infiltrating CD45.2high (APCeFluor780) CD11b2(eFluor 450) CD41(PerCP) CD82(V500) T cells after EAE induction in a WT mouse at Day 15 (Score 4) is shown. Data are representative of three independent experiments.
624
Transcription Factor Staining with Retention of Fluorescent Proteins
Figure 2. Fixation in 1% PFA partially rescues cytosolic fluorescent proteins but inhibits efficient transcription factor staining. All samples were stained IC using eBio-Perm buffer. (a) CD41(PerCP) CD82(V500) splenocytes of a CD4/EYFP mouse are shown. Upper row: EYFP fluorescence after eBio-Fix/Perm fixation. Lower row: EYFP fluorescence after 1% PFA fixation for different fixation times (shaded histograms gated on CD42(PerCP) CD82(V500) cells show background). (b) Statistic of stain index (Si) for EYFP fluorescence as shown in (a). (c) CD90.21(PB) CD41(PerCP) CD82(V500) splenocytes of a WT mouse are shown. Upper row: CD4(PerCP) vs. FoxP3(APC) after eBio-Fix/Perm fixation. Lower row: CD4(PerCP) vs. FoxP3(APC)-positive cells after fixation in 1% PFA for different fixation times. (d) Graphs of percentages as shown in (c). Data are representative of two independent experiments.
Figure 3. Fixation using 2% FA retains cytosolic fluorescent proteins in the cells and allows efficient transcription factor staining. Stainings of either native, eBio-Fix/Perm fixed cells (60 min) or of cells after fixation for different times with 2% FA are shown. (a) FoxP3(eFluor 450) vs. EYFP in CD90.21(APCeFluor780) CD41(PerCP)CD82(V500) splenocytes of a CD4/EYFP mouse. For comparison, the first plot on the left shows forward scatter area (FSC-A) vs. EYFP on native cells. (b) T-bet(eFluor660) vs. EYFP in CD90.21(APCeFluor780) CD42(PerCP) CD81(V500) splenocytes of CD4/EYFP. (c) RORct(APC) expression in CD90.21(PB) CD41(PerCP)/CD81(V500) DP thymocytes of a WT mouse [shaded histograms gated on CD90.21(PB) CD41(PerCP) CD82(V500) SP thymocytes cells show background staining]. Data are representative of two independent experiments.
Original Article
Figure 4. Staining of in vivo generated inflammatory T-cell populations. (a–c, e and f) Gated CD90.21(APCeFluor780) CD41(V500) CD82(PE-Cy7) T cells, (d and g) CD90.21(APCeFluor780) CD42(V500) CD81(V450) T cells of draining LNs from MOG-CFA immunized CD4/ EYFP mice. (a) Histogram showing EYFP fluorescence in native CD41 T cells [shaded histogram gated on CD90.22(APCeFluor780) CD41(PerCP) CD82(V500) cells demonstrates background staining]. (b and c, e and f) CD41 T cells and (d and g) CD81 T cells are stained using the protocol as shown in Figure 5. Different combinations of cytokines vs. TF and EYFP are presented as indicated. (h–l) Gated CD45.2high(APCeFluor780) CD11b2(PE-Cy7) CNS-infiltrating T cells from MOG-CFA-immunized CD4/RFP mice with EAE. (h) Histogram showing RFP fluorescence in native CD41 T cells [shaded histogram gated on CD45.22(APCeFluor780) CD11b2(PE-Cy7) cells demonstrates background staining]. (i–l) Cells stained using the protocol as shown in Figure 5. (i) Histogram shows a certain loss of RFP fluorescent intensity when compared with native cells but the same percentage of tdRFP-positive CD41 T cells [shaded histogram gated on CD45.2low (APCeFluor780) CD11b2(PE-Cy7) cells demonstrates background staining]. (j) Gating of CD41(BV510) tdRFP1 T cells for presentation of IL17A(eFluor450) vs. RORct(PerCPeFluor710) as shown in (k). (l) Quadrants QI–III shown in dot-plot in (k) were used for further gating.
CD41 T cells (neither fixed nor permeabilized) were RFP positive, and fixation with 2% FA completely rescued FPs (Fig. 4i). Using the advantage of our protocol, we could now gate on CD41 RFP1 cells (Fig. 4j) and analyze these cells for cytokine and TF expression (Figs. 4k and 4l). Interestingly, we found during this early stage of EAE development that T-bet expression correlated with expression of RORct and IL-17A, with the highest expression of T-bet found in RORct1/IL626
17A1 CNS-infiltrating T cells (Fig. 4l). This phenomenon may explain the plasticity of Th17 cells in the CNS (2).
DISCUSSION We herein describe a new and simple method allowing staining of TFs and cytokines simultaneously while retaining fluorescence of reporter proteins. Our method combines Transcription Factor Staining with Retention of Fluorescent Proteins
Original Article
Figure 5. Bench-top protocol for transcription factor and cytokine staining with fluorescent reporter protein retention.
fixation in 2% FA for 40–60 min followed by a permeabilization step and staining/washing steps in commercial eBioPerm buffer (Fig. 5). Using this method, FPs are fixed and retained in the cytosol of the cell. Thereby, fluorescent reporter proteins are not washed out completely during permeabilization/staining in subsequently used eBio-Perm buffer during the following TF staining procedure and they can readily be detected by flow cytometry. By defining a proper fixative with the proper timing for fixation, we succeeded in finding a compromise allowing TF staining under fixation conditions strong enough to maintain cytosolic FPs inside of cells. A previous report similarly presented a method for staining TFs with maintenance of reporter proteins (9). This group defined low PFA concentrations (1–2%) as prefixation step before using the commercial eBio-Fix/Perm and eBio-Perm buffers for TF staining. We also tested 1% PFA for varying times as fixative before permeabilization (without using Fix/Perm from eBioscience) and failed to find a condition with effective retention of fluorescence and reliable FoxP3 staining. Additionally, omitting the Fix/Perm step turns our protocol to be easier, and we believe that usage of FA instead of PFA allows a more subtle compromise in fixation. Furthermore, we additionally demonstrate retention of RFP with simultaneous staining of the TF RORct, which proved to be more difficult to stain than FoxP3. Nevertheless, the conclusions of both reports are similar in that a compromise needs to be defined between a strong enough fixation for FP retention which is weak enough not to hamper nuclear protein detection by immunostaining. Despite keeping the percentage of fluorescent reporter positive cells stable, any kind of fixation conditions used in this study showed a certain loss of fluorescence intensity. This
Cytometry Part A 85A: 621627, 2014
is probably at least partially due to fixation of the FP, which might influence the structure or function of the chromophore as described previously (10) and as shown here. Additionally, a further loss of fluorescence protein might occur during the staining steps in permeabilizing buffer even under optimal prefixation conditions (data not shown). Nevertheless, the signal strength surviving the fixation/permeabilization process is in most cases strong enough for its reliable detection by flow cytometry. Leakage from suboptimally fixed cells constitutes the stronger obstacle for detection of FPs in reporter positive cells costained for nuclear proteins. EYFP has a molecular weight which nearly doubles that of the cytokine IFN-c. Therefore, one might speculate why the bigger protein is easier washed out than the smaller cytokine. Although we have no proof for this, our explanation for this differential leakage is that cytokines are not stored in the cytosol where EYFP is located but that they are located in high concentrations in secretory vesicles and on their way to these vesicles in the ER and the Golgi. This additional membrane barrier, together with the densely packed composition of the vesicle lumen, may also retain cytokines under low fixation conditions inside cells. In summary, we provide a new protocol which enables easy and reliable staining of nuclear TFs retaining soluble cytosolic reporter proteins with fluorescence activity.
ACKNOWLEDGMENTS The authors thank Adriana L€angle from the Technical University Darmstadt for technical assistance and Nives H€ ormann from the Center for Thrombosis and Hemostasis (CTH) Mainz for acquisition with the fluorescent reader.
LITERATURE CITED 1. Srinivas S, Watanabe T, Lin CS, William CM, Tanabe Y, Jessell TM, Costantini F. Cre reporter strains produced by targeted insertion of EYFP and ECFP into the ROSA26 locus. BMC Dev Biol 2001;1:4. 2. Kurschus FC, Croxford AL, Heinen AP, W€ ortge S, Ielo D, Waisman A. Genetic proof for the transient nature of the Th17 phenotype. Eur J Immunol 2010;40:3336–3346. 3. Lahl K, Loddenkemper C, Drouin C, Freyer J, Arnason J, Eberl G, Hamann A, Wagner H, Huehn J, Sparwasser T. Selective depletion of Foxp31 regulatory T cells induces a scurfy-like disease. J Exp Med 2007;204:57–63. 4. Lee PP, Fitzpatrick DR, Beard C, Jessup HK, Lehar S, Makar KW, Perez-Melgosa M, Sweetser MT, Schlissel MS, Nguyen S, et al. A critical role for Dnmt1 and DNA methylation in T cell development, function, and survival. Immunity 2001;15:763– 774. 5. Luche H, Weber O, Nageswara Rao T, Blum C, Fehling HJ. Faithful activation of an extra-bright red fluorescent protein in “knock-in” Cre-reporter mice ideally suited for lineage tracing studies. Eur J Immunol 2007;37:43–53. 6. Cardona AE, Huang D, Sasse ME, Ransohoff RM. Isolation of murine microglial cells for RNA analysis or flow cytometry. Nat Protoc 2006;1:1947–1951. 7. Kurschus FC, Pal PP, B€aumler P, Jenne DE, Wiltschi B, Budisa N. Gold fluorescent annexin A5 as a novel apoptosis detection tool. Cytometry Part A 2009;75A:626– 633. 8. Eberl G, Marmon S, Sunshine MJ, Rennert PD, Choi Y, Littman DR. An essential function for the nuclear receptor RORc(t) in the generation of fetal lymphoid tissue inducer cells. Nat Immunol 2004;5:64–73. 9. Grupillo M, Lakomy R, Geng X, Styche A, Rudert WA, Trucco M, Fan Y. An improved intracellular staining protocol for efficient detection of nuclear proteins in YFP-expressing cells. Biotechniques 2011;51:417–420. 10. Ganguly S, Clayton AH, Chattopadhyay A. Fixation alters fluorescence lifetime and anisotropy of cells expressing EYFP-tagged serotonin1A receptor. Biochem Biophys Res Commun 2011;405:234–237.
627
