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Foxp3 fitc

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Foxp3-FITC is a fluorochrome-conjugated antibody that targets the Foxp3 transcription factor. Foxp3 is a key regulator of regulatory T cell (Treg) development and function. The FITC fluorophore allows for the detection and analysis of Foxp3-expressing cells using flow cytometry.

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Lab products found in correlation

Facscalibur, by BD (5 mentions) Cd80 fitc, by BD (4 mentions) Phorbol myristate acetate (pma), by Merck Group (3 mentions) Foxp3 transcription factor staining buffer set, by Thermo Fisher Scientific (3 mentions) Cd4 percp, by BD (3 mentions) Cd25 apc, by BD (3 mentions) Cd4 pe, by BD (3 mentions) Cd25 pe, by Thermo Fisher Scientific (3 mentions) Foxp3 staining buffer set, by Thermo Fisher Scientific (3 mentions) Pd 1 pe, by BioLegend (3 mentions) Cd8 fitc, by BD (3 mentions) Cd4 pe, by Thermo Fisher Scientific (2 mentions) Facs canto 2 instrument, by BD (2 mentions) Efluor 450, by Thermo Fisher Scientific (2 mentions) Cd4 apc cy7, by BD (2 mentions) Cd69 pe, by BD (2 mentions) Fixation permeabilization buffer set, by Thermo Fisher Scientific (2 mentions) Cd25 percp cy5, by BioLegend (2 mentions) Rat anti mouse cd16 cd32 antibody, by BD (2 mentions) Cd3 brilliant violet 510, by BioLegend (2 mentions)

Spelling variants of this product

Anti-Foxp3-FITC (25 citations) FITC-conjugated anti-Foxp3 (7 citations) Foxp3-FITC (FJK-16s, (4 citations) FOXP3-FITC (clone PCH101 (3 citations) Anti-mouse/rat FOXP3-FITC (FJK-16s) (3 citations) FITC anti-mouse Foxp3 (FJK-16S). (2 citations) FITC-labeled anti-Foxp3 (2 citations) FITC)-conjugated anti-mouse Foxp3 (2 citations) Fluorescein isothiocyanate (FITC)-conjugated anti-Foxp3 (2 citations) FITC-conjugated anti-forkhead box P3 (Foxp3), (2 citations)

35 protocols using foxp3 fitc

1

Characterization of Th17 and Treg Cells

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LPLs and pLN cells stimulated for 4 hours with PMA (50 ng/ml; Sigma), ionomycine (1 μg/ml; Sigma), and the Golgi-traffic inhibitor Brefeldin (1 μl/ml; BD Biosciences). Cells were stained with anti-CD3-PE (BD Pharmingen) or anti-CD3-APC (eBioscience) and anti-CD4-APC (Biolegend) or anti-CD4-FITC (BD Pharmingen). Next, the cells were fixed and permeabilized using fixation/permeabilization buffer (eBioscience). For intracellular staining the cells were incubated in permeabilization buffer (eBioscience) containing anti-IL-17-FITC (Biolegend), anti-IFNγ-FITC (BD Pharmingen), anti-IL-4-PE (BD Pharmingen) or Foxp3-FITC (eBioscience). An appropriate isotype matched control antibody was used in all FACS analyses. Cells were analyzed on a FACS Calibur using the CellQuest software (BD Biosciences). Results were analyzed with FlowJo version 7.6.5.
Rogier R., Ederveen T.H., Wopereis H., Hartog A., Boekhorst J., van Hijum S.A., Knol J., Garssen J., Walgreen B., Helsen M.M., van der Kraan P.M., van Lent P.L., van de Loo F.A., Abdollahi-Roodsaz S, & Koenders M.I. (2019). Supplementation of diet with non-digestible oligosaccharides alters the intestinal microbiota, but not arthritis development, in IL-1 receptor antagonist deficient mice. PLoS ONE, 14(7), e0219366.
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2

Flow Cytometry Analysis of Murine Immune Cells

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Animals were sacrificed and the spleens and kidneys were collected for flow cytometry analysis. The monoclonal antibodies used for splenic flow cytometry were CD4-PE, CD3-PE-cy7, Foxp3-FITC, CD69-percp-cy7, B220-PE-cy7, CD21-FITC, CD23-PE, CD11c-PE-cy7, CD11b-APC, F4/80-PE, CD86-FITC, and F4/80-PErCP. The monoclonal antibodies used for renal flow cytometry were CD4-PE, CD3-Percp, Foxp3-FITC, CD45-APC-cy7, CD11b-FITC, CD11c-PE-cy7, F4/80-PE, and Gr-1-Percp (eBioscience, Hanover Park, IL, USA). Cell counting was performed by using a Cellometer® automated cell counting system (Sigma-Aldrich, St Louis, MO, USA) for absolute cell numbers. The Novocyte flow cytometer system (ACEA Bioscience Inc., San Diego, CA, USA) was used for flow cytometry, and analysis was performed as described [11 (link)]. Data were analyzed using Novocyte software (ACEA Bioscience Inc.). At least 200,000 events were acquired for each analysis.
Qin L., Du Y., Ding H., Haque A., Hicks J., Pedroza C, & Mohan C. (2019). Bradykinin 1 receptor blockade subdues systemic autoimmunity, renal inflammation, and blood pressure in murine lupus nephritis. Arthritis Research & Therapy, 21, 12.
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3

Multicolor Flow Cytometry of Skin Immune Cells

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Protocols were conducted as previously described [19 (link)]. CD8α-AF700 (BioLegend), CXCR3-PE (BioLegend), CD4-PE-Cy7 (eBioscience), Foxp3-FITC (eBioscience) and CD45-PB (BioLegend) were used for these studies. Skin samples were prepared as previously described [22 (link)]. Briefly, single cell suspensions (1 million cells) were first incubated with Fc Block (BD Pharmingen. San Diego, CA) for 10 mins, then co-incubated with antibodies for 20 min at 4°C followed by washing with staining buffer (PBS + 1% FBS). Foxp3 intracellular staining was performed using an eBioscience kit (Cat#00-5523-00) according to the manufacturer’s protocol. Flow cytometry was performed on a LSR Fortessa and data were analyzed by FlowJo software (TreeStar).
Pai C.C., Hsiao H.H., Sun K., Chen M., Hagino T., Tellez J., Mall C., Blazar B.R., Monjazeb A., Abedi M, & Murphy W.J. (2014). Therapeutic benefit of bortezomib on acute GVHD is tissue specific and is associated with IL-6 levels. Biology of blood and marrow transplantation : journal of the American Society for Blood and Marrow Transplantation, 20(12), 1899-1904.
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4

Multiparameter Flow Cytometry Immunophenotyping

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Fc-gamma receptors were blocked with a rat anti-mouse CD16/CD32 antibody (BD). Mouse-specific antibodies were the following: CD3-Brilliant violet 510, CD25-PercP Cy5.5, I-A/I-E-Brilliant violet 510, CD45R/B220-PE-Cy7, CD43-APC, CTLA-4-APC (all from BioLegend), CD4-APC-Cy7, CD69-PE, CD11c-PercP Cy5.5, CD19-APC-Cy7, CD40-FITC (all from BD Pharmingen), CD8α-PE-Cy7, FoxP3-FITC, CD86-APC, CD83-FITC, CD80-PE-Cy7, IgM-PercP-eFluor 710 (all from eBioscience).
During the experimental set-up, CD3 antibody was included in the staining panel (BV510 BioLegend clone 17A2) and used for the gating of T cells. The results obtained with gating on CD3+ CD4+ CD8- were similar to those obtained with gating on CD4+ CD8-. Due to the limitation in the maximum number of colours that can be discriminated with the FACS Canto II, CD3 staining was omitted in subsequent stainings.
Dead cells were excluded using the fixable viability dye-eFluor 450 (eBioscience), and intracellular staining was performed using the fixation/ permeabilization buffer set from eBioscience. Flow cytometry measurements were performed on a FACS Canto II instrument (BD) and the data were analyzed with FlowJo software (Tree Star).
Sordé L., Spindeldreher S., Palmer E, & Karle A. (2017). Massive immune response against IVIg interferes with response against other antigens in mice: A new mode of action?. PLoS ONE, 12(10), e0186046.
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5

Phenotypic Analysis of Activated T Cells

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Cultured T cells were stained for surface expression of CD69 and CD101 (both from BD Biosciences, San Diego, CA). Detection of FOXP3+ or IL17-producing cells was determined by intracellular staining. Briefly, cells were stimulated for 4 h with PMA and ionomycin; after 2 h, GolgiStop (BD Biosciences) was added. Stimulated cells were fixed and permeabilized with Transcription Factor Staining Buffer Set (eBioscience, San Diego, CA) stained with anti-IL17-phycoerythrin (BD Biosciences), Foxp3-APC, or FOXP3-FITC (eBioscience) according to manufacturer’s instructions and detected in flow cytometer LSRII (Becton Dickinson, San Jose, CA). Events were collected and analyzed by using flowjo software (Tree Star, Ashland, OR).
Mohammad I., Nousiainen K., Bhosale S.D., Starskaia I., Moulder R., Rokka A., Cheng F., Mohanasundaram P., Eriksson J.E., Goodlett D.R., Lähdesmäki H, & Chen Z. (2018). Quantitative proteomic characterization and comparison of T helper 17 and induced regulatory T cells. PLoS Biology, 16(5), e2004194.
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6

Treg Cell Phenotypic Analysis During Expansion

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Treg cell phenotype was analyzed immediately after thawing, after subsequent overnight culture and during Treg expansion (on days: 0, 7, 13). To measure Treg phenotype, a sample with 2 × 105 cells was taken and processed accordingly to the manufacturer instruction for the FoxP3/Transcription Factor Staining Buffer Set (eBioscience, Inc., San Diego, CA, USA). Subsequently, a sample was stained with the following antibodies: FoxP3 FITC (eBioscience, Inc., San Diego, CA, USA), CD4 PerCP, CD25 APC, CD127 PE (BDBiosciences, San Jose, CA, USA). Data for cell phenotyping was acquired by flow cytometry.
Gołąb K., Grose R., Placencia V., Wickrema A., Solomina J., Tibudan M., Konsur E., Ciepły K., Marek-Trzonkowska N., Trzonkowski P., Millis J.M., Fung J, & Witkowski P. (2018). Cell banking for regulatory T cell-based therapy: strategies to overcome the impact of cryopreservation on the Treg viability and phenotype. Oncotarget, 9(11), 9728-9740.
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7

Hamster-Derived Anti-PD-1 Antibody Protocol

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MH5A was previously developed by immunizing Armenian hamsters with a mouse PD-1HIg fusion protein and Freund’s adjuvant as previously described in detail (22 (link)). Control hamster IgG was purchased from Rockland Immunochemicals (Gilbertsville, PA). Fluorescently labeled antibodies for flow cytometry including CD4, CD8, H-2Kd, IFN-γ, CD19, CD25, Gr-1, CD11b, DX5, and anti-hamster IgG-biotin were purchased from BD Biosciences (San Jose, CA). CD3 and CD28 mAbs for cell culture were also purchased from BD Biosciences. AnnexinV and 7-AAD were purchased from BD Biosciences. FoxP3-APC, FoxP3-FITC, and streptavidin-APC and PerCP, and TNF-α ELISA were purchased from eBioscience (San Diego, CA). Ki67-APC and ZombieNIR were purchased from Biolegend. CFSE (Vybrant CFDA cell tracer kit) was purchased from Life Technologies. TGF-β and rIL-2 were purchased from R&D Systems (Minneapolis, MN).
Flies D.B., Higuchi T, & Chen L. (2015). Mechanistic Assessment of PD-1H Coinhibitory Receptor-Induced T-Cell Tolerance to Allogeneic Antigens. Journal of immunology (Baltimore, Md. : 1950), 194(11), 5294-5304.
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8

Detailed Flow Cytometry Immunophenotyping

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Mouse thymocytes and splenocytes were isolated in RPMI containing 5% fetal calf serum, followed by red blood cell depletion using the ACK lysing buffer (Gibco). Flow cytometric analysis was performed on cells according to a previously described procedure (Jiang et al., 2011 (link)). Antibodies used for analyses: CD3 pacific blue (BD, clone 500A2, 558214), CD8 APC-Cy7 (BD, clone 53-6.7, Cat. No. 557654), CD4 PE (BD, clone GK1.5, Cat. No. 553730), CD80 FITC (BD, Cat. No. 553768), mouse Vβ TCR screen panel (BD, Cat. No. No. 557004), CD25 APC (eBioscience, clone PC61.5, Cat. No. 17-0251-82), Foxp3 FITC (eBioscience, clone FJK16a, Cat. No. 11-5773-82), CD3e FITC (eBioscience, clone 145-2C11, 11-0031-82), CD274 PE (eBioscience, B7-H1, PD-L1, clone MIH5, 12-5982-81), ly-6G (Gr-1) Alexa Fluor 700 (eBioscience, clone RB6-8c5, 56-5931-80), CD11b PE-Cy7 (eBioscience, clone M1/70, 25-0112-81), CD11c 780 (eBioscience, clone N418, 47-0114-80), CD49f (intergrin alpha 6) PE-Cyanine 7 (eBioscience, clone GOH3, Cat. No. 25-0495-80), CD45.1 PE-Cy7 (eBioscience, clone A20, 25-0453-82), CD45.2 eFluor 450 (eBioscience, clone 104, Cat# 48-0454-82), and FITC conjugated UEA1 (Vector, Cat. No. FL-1061). Labeled cells were analyzed on a LSR-II Flow Cytometer (Becton Dickinson). Data were analyzed using FlowJo software.
Zhu F., Willette-Brown J., Song N.Y., Lomada D., Song Y., Xue L., Gray Z., Zhao Z., Davis S.R., Sun Z., Zhang P., Wu X., Zhan Q., Richie E.R, & Hu Y. (2017). Autoreactive T Cells and Chronic Fungal Infection Drive Esophageal Carcinogenesis. Cell host & microbe, 21(4), 478-493.e7.
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9

Flow Cytometry Analysis of Mouse and Human T Cells

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Anti-human CD3e-V500, CD4-APC-H7, CD8-APC-H7, CD25-PE, CD39-PE (clone TU66), CD73-APC (clone AD2), PD1-PE-Cy7 (clone EH12.1), and CD127-PerCP-Cy5.5 antibodies were purchased by BD Biosciences (Germany), anti-human FOXP3-FITC antibody was from eBioscience (Germany). Anti-mouse CD3e-PerCP-Cy5.5, -APC, -V500 and -V450; CD4-APC, -BV421, -APC-Cy7 and -APC-H7; CD8a-APC-Cy7, -PE-Cy7 and -APC-H7; CD25-APC, -APC-Cy7 and -V450; CD44-PE; CD45-V500; CD45RB-PE; CD62 L-APC; CD69-APC; CD73-BV605 (clone TY/11.8) and PD1-PerCP-Cy5,5 (clone J43) were purchased by BD Biosciences (Germany). Purified anti-mouse CD3e and CD28, as well as FOXP3-FITC, CD73- eFluor® 450, -PE (clone TY/11.8 for both) and CD39-PE-Cy7, -PE (clone DM24S1 for both) were from eBioscience (Germany). BSA, Trypan blue, PMA, and Ionomycin were obtained from Sigma (Germany). Phosphate buffer saline (PBS), RPMI-1640 with L-glutamine and Penicillin-Streptomycin were purchased by PAA (Germany). Fetal bovine serum (FBS) was from PAN Biotech (Germany). Brefeldin A was purchased by BD Biosciences (Germany). Foxp3/Transcription Factor Staining Buffer Set and Permeabilization Buffer (10X) were obtained from eBioscience (Germany). Red Blood Cell (RBC) Lysis Buffer was from Biolegend (Germany).
Shevchenko I., Mathes A., Groth C., Karakhanova S., Müller V., Utikal J., Werner J., Bazhin A.V, & Umansky V. (2020). Enhanced expression of CD39 and CD73 on T cells in the regulation of anti-tumor immune responses. Oncoimmunology, 9(1), 1744946.
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10

Comprehensive Immune Cell Profiling in Sepsis

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Mice were sacrificed and spleens were harvested at 24h after CLP. Splenocytes were stained with anti-CD3-Alexa 700 (BD), anti-CD4-PB (Biolegend, clone RM4–5), anti-CD8-PO (Invitrogen, clone MCD0830), anti-CD44-PerCP (Biolegend, clone IM7), anti-CD62L-PE (BD), anti-CD28-PE-Cy7 (Biolegend, clone E18) and anti-CD25-APC-Cy7 (BD). For detection of cell apoptosis, splenocytes were stained with a FITC Annexin V apoptosis detection kit with 7-AAD (Biolegend). Anti-Bcl-xL (54H6) and Bcl-2 (Biolegend, clone BCL/10C4) were used to detect engagement of the mitochondrial pathway of apoptosis while anti-CD95 (Biolegend, clone DX2) and anti-TNFR Type Ⅰ (Biolegend, clone 55R-286) were stained to detect expression of death receptors on T cells. Cells were intracellularly stained with anti-Ki-67 (Biolegend, clone 16A8) to assay for cell proliferation. Tregs were identified via intracellular staining for Foxp3-FITC (Ebioscience, clone FJK-16S) using the Foxp3 staining kit (Ebioscience). B cells were stained with anti-CD19-FITC (Biolegend). NK cells were stained with anti-NK1.1-PE (Ebioscience). Dendritic cells were stained with anti-CD11c-PE-Cy7 (BD). Neutrophils were stained with anti-Gr-1-Alexa 700 (Biolegend) and anti-CD11b-PerCP (Biolegend). Accucheck Counting Beads (Thermo Fisher Scientific) were added after staining to calculate the absolute number of cells per spleen.
Sun Y., Xie J., Anyalebechi J.C., Chen C.W., Sun H., Xue M., Liang Z., Morrow K.N., Coopersmith C.M, & Ford M.L. (2020). CD28 agonism improves survival in immunologically experienced septic mice via IL-10 released by Foxp3+ regulatory T cells. Journal of immunology (Baltimore, Md. : 1950), 205(12), 3358-3371.
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