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Foxp3 transcription factor staining buffer kit

Manufactured by Cytek Biosciences
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The Foxp3/Transcription Factor Staining Buffer Kit is a laboratory product designed for the intracellular staining and detection of Foxp3 and other transcription factors. The kit provides the necessary buffers and reagents to facilitate the fixation, permeabilization, and staining of cells, enabling the analysis of transcription factor expression using flow cytometry or other techniques.

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Lab products found in correlation

Ghost dye red 780, by Cytek Biosciences (3 mentions) Paraformaldehyde (pfa), by Thermo Fisher Scientific (2 mentions) Tnb 1213 l150, by Cytek Biosciences (2 mentions) Rm4 5, by Cytek Biosciences (2 mentions) Ebio4b10, by Thermo Fisher Scientific (2 mentions) Rpa t4, by Cytek Biosciences (2 mentions) Rpa t8, by Cytek Biosciences (2 mentions) Mp4 25d2, by Cytek Biosciences (2 mentions) Foxp3 fix perm kit, by Thermo Fisher Scientific (2 mentions) Anti mouse cd16 32 clone 13, by BioLegend (2 mentions) Live dead fixable green, by Thermo Fisher Scientific (2 mentions) Live dead fixable aqua, by Thermo Fisher Scientific (2 mentions) Pe anti human cd68, by BioLegend (2 mentions) Viability stain, by Thermo Fisher Scientific (2 mentions) Apc faser kit, by Miltenyi Biotec (2 mentions) Accuri c6, by BD (2 mentions) Fc block, by BD (2 mentions) Lsrfortessa, by BD (2 mentions) 4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions) Celltrace violet, by Thermo Fisher Scientific (2 mentions)

34 protocols using foxp3 transcription factor staining buffer kit

1

Intracellular Cytokine Staining for T Cells

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Intracellular cytokine staining was performed with the Foxp3/Transcription Factor Staining Buffer Kit (Tonbo) per manufacturer’s instructions. Briefly, T cells were mixed with 2x106 C57BL/6 splenocytes and stimulated with 0.5 μg/mL of TAG epitope I peptide in cRPMI for 4 hours at 37°C in the presence of brefeldin A (BioLegend). Where indicated, stimulation media contained anti-CD107a antibody. Following peptide stimulation, cells were stained with for surface markers, fixed, permeabilized, and stained for IFNγ, TNF, perforin, and granzyme B. Intracellular transcription factor staining was performed with the Foxp3/Transcription Factor Staining Buffer Kit (Tonbo) per manufacturer’s instructions
Rudloff M.W., Zumbo P., Favret N.R., Roetman J.J., Detrés Román C.R., Erwin M.M., Murray K.A., Jonnakuti S.T., Dündar F., Betel D, & Philip M. (2023). Hallmarks of CD8 T cell dysfunction are established within hours of tumor antigen encounter prior to cell division. Nature immunology, 24(9), 1527-1539.
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2

Cytotoxic PBMC Activation Assay

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Human PBMCs (5 × 105/well) were seeded in a 24-well cell culture plate. To achieve sub-optimal activation of PBMCs, polyinosinic–polycytidylic acid (poly I:C) (10 µg/ml) was added to the media. After 18 hr incubation, MDA-MB-231-LM2 cells (2.5 × 105/well) were added to the PBMCs. Subsequently, isotype IgG control, Tw1S4_6 or Tw1S4_AM6 antibodies (200 nM) were added to the media and anti-CD107a antibody was added directly into the media. After 1 hr, brefeldin A solution (Biolegend) was added to the media. Cells were collected after 3 hr and stained with anti-CD3 and anti-CD56 antibodies. Intracellular staining of interferon gamma (IFN-ɣ) and granzyme B were conducted according to the manufacturer’s protocol (Foxp3/Transcription Factor Staining Buffer Kit, Tonbo bioscience). Stained cells were analyzed by flow cytometry (LSRFortessa H0081, BD bioscience).
Kalscheuer S., Khanna V., Kim H., Li S., Sachdev D., DeCarlo A., Yang D, & Panyam J. (2019). Discovery of HSPG2 (Perlecan) as a Therapeutic Target in Triple Negative Breast Cancer. Scientific Reports, 9, 12492.
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3

Comprehensive Cytokine Profiling with Flow Cytometry

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Surface staining was carried out using PBS with 1% FBS. For intracellular staining of cytokines, cells were first surface stained, then fixed with 1% paraformaldehyde (catalog no. 19943; Affymetrix, Santa Clara, CA) for 15 min at room temperature and permeabilized in permeabilization buffer (TNB-1213-L150; Tonbo Biosciences) before staining for cytokines. Staining for transcription factors was performed with the Foxp3/Transcription Factor Staining Buffer Kit (TNB-1022-L160; Tonbo Biosciences) according to the manufacturer’s instructions. Flow cytometry data were collected with CytoFLEX (BD Biosciences, Franklin Lake, NJ), and results were analyzed using FlowJo software (Becton Dickinson, Franklin Lakes, NJ). Abs specific for mouse CD4 (RM4–5), CD8 (53–6.7), and IL-4 (11B11) were purchased from Tonbo Biosciences; and those for IFN-γ (4S.B3), TNF (MAB11), GATA3 (L50–823), and Tbet (eBio4B10) were purchased from eBioscience (San Diego, CA). Abs specific for human CD4 (RPA-T4), CD8 (RPA-T8), and IL-4 (MP4–25D2) were purchased from Tonbo Biosciences; and those for IFN-γ (4S.B3), GATA3 (16E10A23), and Tbet (eBio4B10) were obtained from eBioscience.
Poudel B., Yorek M.S., Mazgaeen L., Brown S.A., Kanneganti T.D, & Gurung P. (2020). Acute IL-4 Governs Pathogenic T Cell Responses during Leishmania major Infection. ImmunoHorizons, 4(9), 546-560.
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4

Intracellular Cytokine Staining of CD4+ T Cells

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The in vitro differentiated CD4+ T cells were stimulated using an immobilized anti-TCR-β mAb (3 µg/ml, H57–597, BioLegend) for 6 h in the presence of monensin (2 µM). The cells were fixed with 4% paraformaldehyde (Cat#163–20145, Wako) and permeabilized with permeabilization buffer (50 mM NaCl, 5 mM EDTA, 0.02% NaN3, and 0.5% Triton X-100). Then, the cells were stained using the following antibodies, anti-IL-4-PE (Cat#504103, BioLegend, 1:50), anti-IL-5-APC (Cat#504305, BioLegend, 1:50), anti-IL-13-PE (Cat#12–7133–41, eBiosience, 1:50), anti-IFN-γ-FITC (Cat#562019, BD Biosciences, 1:500), and anti-IL-2-APC (Cat#503809, BioLegend, 1:50). For the intracellular staining of Gata3, the Foxp3/Transcription Factor Staining Buffer Kit (cat#TNB-0607, TONBO) was used according to the manufacturer’s protocol. Flow cytometry was performed using a Gallios Flow Cytometer instrument (Beckman Coulter) and a FACS Caliber instrument (BD Biosciences) and the results were analysed using the FlowJo software program (Tree Star, Ashland, OR, USA).
Nomura S., Takahashi H., Suzuki J., Kuwahara M., Yamashita M, & Sawasaki T. (2019). Pyrrothiogatain acts as an inhibitor of GATA family proteins and inhibits Th2 cell differentiation in vitro. Scientific Reports, 9, 17335.
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5

Detailed Immune Cell Phenotypic Analysis

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Cell suspensions were first stained for surface markers (such as CD45.2 and CD8) and then fixed and stained for TCF1 (clone C63C9), T-bet (clone 4B10), Eomes (clone Dan11mag), Blimp1 (clone 5E7), or Ki67 (clone B56) using the Foxp3/Transcription Factor Staining Buffer Kit (Tonbo Biosciences) according to manufacturer instructions. To assess IFN-γ and TNF production, live cell suspensions were stimulated by plate bound anti-CD3ε (1 μg/ml coating concentration; 145-2C11; BioXCell) and anti-CD28 (1 μg/ml coating concentration; 37.51; BioXCell) or PMA and Ionomycin (20 ng/ml and 1 μg/ml, respectively, both from Sigma-Aldrich) for 2 hours at 37 °C, after which GolgiPlug was added, and the cells incubated for an additional 4 hours in 37 °C. After staining for surface markers, samples were fixed with IC Fixation Buffer (eBioscience), treated with permeabilization buffer (eBioscience), and stained with fluorescently labeled anti-IFN-γ (clone XMG1.2) and anti-TNF (clone MP6-XT22) antibodies, followed by flow cytometry analysis (BD Biosciences LSRII and Beckman Coulter Cytoflex LX). Details pertaining to antibody staining, including clone, fluorophore, and dilution, are provided in table S2.
Zhang M., Zhao Z., Pritykin Y., Hannum M., Scott A.C., Kuo F., Sanghvi V., Chan T.A., Seshan V., Wendel H.G., Schietinger A., Sadelain M, & Huse M. (2021). The miR-200c–EpCAM axis reprograms therapeutic T cells to enhance anti-tumor immunity in mouse models. Science translational medicine, 13(611), eabg4328.
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6

Splenocyte stimulation and intracellular staining

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In some experiments, splenocytes were stimulated ex vivo after isolation from infected mice. Splenocytes were incubated with Trp2 peptide (10−6 M) and Golgiplug (BD Biosciences) for 4–6 hr at 37°C; parallel wells with no peptide were used as a control. Cells were washed and stained with surface antibodies, followed by fixation and permeabilization with a FoxP3 Fix/Perm kit (eBioscience) or FoxP3/transcription factor staining buffer kit (Tonbo Biosciences) and staining with intracellular antibodies.
Truckenbrod E.N., Burrack K.S., Knutson T.P., Borges da Silva H., Block K.E., O'Flanagan S.D., Stagliano K.R., Hurwitz A.A., Fulton R.B., Renkema K.R, & Jameson S.C. (2021). CD8+ T cell self-tolerance permits responsiveness but limits tissue damage. eLife, 10, e65615.
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7

Evaluating CD8+ T-cell Responses

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LCL pulsed with WT1 peptide at 0, 1, and 100 nM were used as target cells. 1 × 105 cells of target and effector cells were co-cultured for 4 h in the presence of monensin (GolgiStop; BD Biosciences) in 96-well round bottom plate. Cells were stained with antibody against CD8a, followed by performing the intracellular staining with anti-IFNγ antibody using Foxp3/Transcription Factor Staining Buffer Kit (Tonbo Biosciences).
Maeda T., Nagano S., Kashima S., Terada K., Agata Y., Ichise H., Ohtaka M., Nakanishi M., Fujiki F., Sugiyama H., Kitawaki T., Kadowaki N., Takaori-Kondo A., Masuda K, & Kawamoto H. (2020). Regeneration of Tumor-Antigen-Specific Cytotoxic T Lymphocytes from iPSCs Transduced with Exogenous TCR Genes. Molecular Therapy. Methods & Clinical Development, 19, 250-260.
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8

Multiparameter Flow Cytometry Analysis

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Cultured cells and primary T cells were stained with the indicated Abs. Data were obtained with Gallios (Beckman Coulter) and analyzed using FlowJo (Tree Star). Abs against Bcl6 (K112-91) and CXCR5 (2G8) were purchased from BD bioscience. Abs against CD3 (145-2C11), CD80 (16-10A-1), CD86 (GL-1), and PD-L2 (TY25) were purchased from eBioscience. The Ab against CD8 (5H10) was purchased from Thermo Fisher Scientific. Abs against CD3 (17A2), CD4 (RM4-5), CD28 (37.5), CD45 (30-F11), ICOS (C398.4A), ICOSL (HK5.3), PD-1 (RMP1-30), and PD-L1 (10F.9G2), Brilliant violet 421-conjugated streptavidin, armenian hamster IgG (HTK888), rat IgG2b (RTK4530), and syrian hamster IgG (SHG-1) were purchased from Biolegend. FoxP3 Transcription Factor Staining Buffer Kit (TONBO) was used for the staining of Bcl6.
Mizuno R., Sugiura D., Shimizu K., Maruhashi T., Watada M., Okazaki I.M, & Okazaki T. (2019). PD-1 Primarily Targets TCR Signal in the Inhibition of Functional T Cell Activation. Frontiers in Immunology, 10, 630.
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9

Comprehensive Immune Cell Profiling

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FACS analysis was performed on an Attune NxT cytometer (Thermo Fisher) and analyzed with FlowJo (TreeStar). Experiments were acquired live or fixed (BD Cytofix/Cytoperm Fixation and Permeabilization kit (BD Biosciences)). Bcl-xl staining used the Foxp3/Transcription Factor Staining Buffer kit (Tonbo). All analyses included size exclusion (forward scatter [FSC] area/ side scatter [SSC] area), doublet exclusion (FSC height/FSC area), and dead cell exclusion (Ghost Dye Red 780, Tonbo). Antibodies used were: CD4 (GK1.5 or RM4–5), CD8α (53–6.7 or 2.43), CD11b (M1/70), CD45.2 (104), CD45.1 (A20), Bcl-xl (7B2.5), P2X7 receptor (polyclonal, Enzo Life Sciences), RORγt (AFKJS-9), B220 (RA3–62B), CD19 (6D5), CD11c (N418), NK1.1 (PK136), Gr-1 (RB6–8C5), Ter119 (TER-119), and TCRβ (H57–597).
Philips R.L., McCue S.A., Rajcula M.J, & Shapiro V.S. (2019). HDAC3 protects DP thymocytes from P2X7 receptor-induced cell death. Journal of immunology (Baltimore, Md. : 1950), 202(4), 1033-1038.
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10

Comprehensive Immune Cell Profiling

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Briefly, cells were incubated with an Fc-blocking antibody (clone 2.4G2) at 4° C for 15 min, prior to surface staining. Antibodies to IgA (eBioscience; clone mA-6E1), IgG1 (BioLegend; clone RMG11), IgG2b (BioLegend; clone RMG2b-1), CD4 (BioLegend; clone GK1.5), CD8α (BioLegend; clone 53–6.7), CD19 (BioLegend; clone 6D5), B220 (BioLegend; clone RA3–6B2), TCRβ (BioLegend; clone H57–597), CD25 (BioLegend; clone 3C5) were used with viability determined using a Zombie Aqua™ Fixable Viability Kit (Biolegend). Treg cell staining was conducted using Tonbo Biosciences buffers (Foxp3/Transcription Factor Staining Buffer Kit; cat no: TNB0607-KIT). Cells were stained as above, then fixed for 1 h at RT prior to permeabilzation. The cells were then incubated with the 2.4G2 Fc-blocking antibody at 4° C for 15 min prior to staining with antiFoxP3 PE (eBioscience; clone NRRF-30) for 30 min at 4° C. Hybridoma supernatants containing mAbs used for cell purification or stimulation, were generously provided by the late Charles Janeway Jr. (Yale University). RPMI-1640 media and heat-inactivated FCS were purchased from Invitrogen and Gemini, respectively.
Li Y.Y., Pearson J.A., Chao C., Peng J., Zhang X., Zhou Z., Liu Y., Wong F.S, & Wen L. (2017). Nucleotide-binding oligomerization domain-containing protein 2 (Nod2) modulates T1DM susceptibility by gut microbiota. Journal of autoimmunity, 82, 85-95.
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