Foxp3 apc fjk 16s

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FoxP3 APC (FJK-16s) is a fluorescence-conjugated antibody that binds to the FoxP3 transcription factor, a marker for regulatory T cells. This product is designed for use in flow cytometry applications to identify and quantify regulatory T cells in biological samples.

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16 protocols using foxp3 apc fjk 16s

Analysis of Tregs and MSCs in Lung Lymph Nodes

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For the analysis of Tregs and MSCs in lung lymph nodes, the pulmonary hilar lymph nodes were collected and teased apart into a single cell suspension by pressing with the plunger of a 3 ml syringe. Harvested lung tissues were minced and incubated for 45 min at 37°C in DMEM supplemented with 10ug/ml DNase I and 1 mg/ml collagenase D (Sigma Aldrich) in a shaking water bath. After that, the digested lung tissue was passed through a 70-um nylon strainer (BD Biosciences) to obtain single cell suspensions. Red blood cells were lysed by ACK lysis buffer. For Tregs, the cells were first stained with anti-CD4-FITC (RM4–5, eBioscience) and anti-CD25-PE antibodies (PC61.5, eBioscience), followed by intracellular staining with FoxP3-APC (FJK-16s, eBioscience) or APC-conjugated rat IgG2a isotype control (eBioscience). For MSCs, cells were stained with Sca-1, CD29, CD45, and CD11b fluorochrome conjugated antibodies (eBioscience). These samples were analyzed on a FACSCalibur flow cytometer (BD Biosystems).

Xu T., Zhou Y., Qiu L., Do D.C., Zhao Y., Cui Z., Wang H., Liu X., Saradna A., Cao X., Wan M, & Gao P. (2015). Aryl Hydrocarbon Receptor Protects Lungs from Cockroach Allergen Induced Inflammation by Modulating Mesenchymal Stem Cells. Journal of immunology (Baltimore, Md. : 1950), 195(12), 5539-5550.

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Multiparameter Flow Cytometry Analysis

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Flow cytometry analyses were carried out using a three-laser configuration Thermo Fisher Scientific Attune Nxt flow cytometry system. Data were analyzed using the FlowJo software (TreeStar). The gates were set using the fluorescence minus one (FMO) control strategy. FMO controls are samples that include all conjugated Abs present in the test samples except one. The channel in which the conjugated Ab is missing is the one for which the FMO provides a gating control. The following mAbs were used: CD3 PerCP-Cy5.5 (145-2C11, eBioscience, San Diego, CA, USA); CD4 APC-eFluor 789 (GK1.5, eBioscience, San Diego, CA, USA); CD8 Super Bright 702 (53-6.7, Invitrogen, Carlsbad, CA, USA); CD11b FITC (M1/70, BioLegend); Gr1 BV510 (RB6-8C5, BioLegend); CD11c Pe-Cyanine7 (N418, eBioscience, San Diego, CA, USA); CD206 PE (MR6F3, Invitrogen, Carlsbad, CA, USA); FoxP3 APC (FJK-16s, eBioscience, San Diego, CA, USA) and IL-10 Alexa Fluor 700 (JES5-16E3, eBioscience, San Diego, CA, USA).

Biagioli M., Carino A., Di Giorgio C., Marchianò S., Bordoni M., Roselli R., Distrutti E, & Fiorucci S. (2020). Discovery of a Novel Multi-Strains Probiotic Formulation with Improved Efficacy toward Intestinal Inflammation. Nutrients, 12(7), 1945.

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Comprehensive Flow Cytometry Analysis

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Flow cytometry analyses were carried out using a two-laser standard configuration BD FACSVia™ flow cytometry system. We analyzed 6–10 mice for each experimental group. Data were analyzed using FlowJo software (TreeStar, Ashland, OR, USA). The gates were set using fluorescence minus one (FMO,) control strategy. FMO controls are samples that include all conjugated Abs present in the test samples except one. The channel in which the conjugated Ab is missing is the one for which the FMO provides a gating control. The following mAbs were used: CD4 PerCp-Cy5.5 (RM4-5, eBioscience, San Diego, CA, USA); CD11b Pe-Cy7 (M1/70, eBioscience); Gr1 PE (RB6-8C5, BioLegend); IL-10 FITC (JES5-16E3, eBioscience) and FoxP3 APC (FJK-16s, eBioscience).

Biagioli M., Capobianco D., Carino A., Marchianò S., Fiorucci C., Ricci P., Distrutti E, & Fiorucci S. (2019). Divergent Effectiveness of Multispecies Probiotic Preparations on Intestinal Microbiota Structure Depends on Metabolic Properties. Nutrients, 11(2), 325.

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Flow Cytometric Analysis of Regulatory T Cells

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The following anti-human antibodies were used: CD4-PE/Cy5, CD25-FITC, and Foxp3-Alexa647 from BioLegend (San Diego, CA, USA). Mouse cells were stained with fluorochrome-labeled mAbs to CD4-Pacific Blue, Brilliant Violet 605 and Alexa-700 (RM4-5), CD8-Pacific Blue (53-6.7), CD25-APC (7D4), Ki-67-PE (Ki-67), and Thy1.1-PercP (Ox-7) from BioLegend. Foxp3-APC (FJK-16s), dead cell marker Viability Dye eFluor™ 780, and staining reagents from eBioscience (San Diego, CA, USA) were used according to the manufacturer’s instructions. To analyze expression of surface proteins, the cells were stained with the appropriate antibodies for 20 min at 4°C, washed once with FACS buffer (PBS, 0.1% BSA, and 0.02% NaN₃), and fixed with 2% paraformaldehyde. For intracellular staining of Foxp3 and Ki-67, the cells were first surface stained, permeabilized with Fix/Perm (eBioscience), and stained with Foxp3 and Ki-67 antibodies diluted in Perm/Wash (eBioscience). To calculate absolute Treg numbers, unlabeled microbeads (BD Biosciences, Franklin Lakes, NJ, USA) were added to the stained cells and the following formula was used: absolute Treg numbers = (beads used × Treg events)/beads measured. Acquisition was performed on a BD™ LSR II or FACSCalibur, and data were analyzed using FlowJo software (TreeStar Inc., Ashland, OR, USA).

Langenhorst D., Tabares P., Gulde T., Becklund B.R., Berr S., Surh C.D., Beyersdorf N, & Hünig T. (2018). Self-Recognition Sensitizes Mouse and Human Regulatory T Cells to Low-Dose CD28 Superagonist Stimulation. Frontiers in Immunology, 8, 1985.

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Tregs and TβRI/II Analysis in Lung and MSCs

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For the analysis of Tregs in lung lymph nodes, the pulmonary hilar lymph nodes were collected and teased apart into a single cell suspension by pressing with the plunger of a 3 ml syringe. The cells were first stained with anti-CD4-FITC(RM4-5, eBioscience, San Diego, CA, USA) and anti-CD25-PE antibodies (PC61.5, eBioscience), followed by intracellular staining with FoxP3-APC(FJK-16s ,eBioscience) or APC-conjugated rat immunoglobulin (Ig) G2a isotype control (eBioscience) using a FoxP3 staining kit (eBioscience). The samples were then analyzed on a FACSCalibur flow cytometer (BD Biosystems). Similar approaches were used for the analysis of TβRI and TβRII (Santa Cruz Biotechnology Inc.) in bone morrow derived MSCs.

Gao P., Zhou Y., Xian L., Li C., Xu T., Plunkett B., Huang S.K., Wan M, & Cao X. (2014). Functional Effects of TGF-beta1 on Mesenchymal Stem Cell Mobilization in Cockroach Allergen Induced Asthma. Journal of immunology (Baltimore, Md. : 1950), 192(10), 4560-4570.

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Tumor Cell Isolation and Analysis

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Tumors were minced, incubated in 100 units/mL collagenase IV (Thermo Fisher Scientific, Waltham, MA) and RPMI supplemented with 10% FBS for 30 minutes at 37°C in a Stomacher machine set at normal speed, washed through 40-micron nylon cell strainers (Falcon; BD Biosciences) in PBS with 2% FBS. After washing, cells were lysed using 1X BD Pharm Lyse Buffer (BD Biosciences). Cells were immediately stained and subsequently analyzed by flow cytometry (Live/Dead-PacOrange, Invitrogen; CD4-PE, H129.19, BD Biosciences; CD3-AF488, 145-2C11, BD Biosciences; CD8-PerCP-Cy5.5, 53–6.7, BD Biosciences; FOXP3-APC, FJK-16 s, eBioscience; EGFRvIII-specific CAR-AlexaFluor 647 multimer). Intracellular staining was performed for FOXP3 using FOXP3 Transcription Factor Staining Buffer Kit (ThermoFisher Scientific; Invitrogen).

Suryadevara C.M., Desai R., Abel M.L., Riccione K.A., Batich K.A., Shen S.H., Chongsathidkiet P., Gedeon P.C., Elsamadicy A.A., Snyder D.J., Herndon JE I.I., Healy P., Archer G.E., Choi B.D., Fecci P.E., Sampson J.H, & Sanchez-Perez L. (2018). Temozolomide lymphodepletion enhances CAR abundance and correlates with antitumor efficacy against established glioblastoma. Oncoimmunology, 7(6), e1434464.

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Analyzing Regulatory T Cells After BMT

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On day 14 after allogeneic bone marrow transplantation, splenocytes were stained with CD4 PE-Cy5 (H129.19), H₂K^b PE (AF6.88.5), CD3 FITC (145-2C11) and FoxP3 APC (FJK.16s; eBioscience). After secondary transfer to Rag2^−/− recipients, splenic T cells were isolated and intracellular flow cytometry was performed as outlined in the Online Supplementary Methods.

Amarnath S., Laurence A., Zhu N., Cunha R., Eckhaus M.A., Taylor S., Foley J.E., Ghosh M., Felizardo T.C, & Fowler D.H. (2017). Tbet is a critical modulator of FoxP3 expression in autoimmune graft-versus-host disease. Haematologica, 102(8), 1446-1456.

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Multicolor Flow Cytometry of Immune Cells

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Single cell suspensions were prepared from spleen, colon LP, and mesenteric lymph nodes, and stained on ice using predetermined optimal concentrations of each antibody (Ab) for 20–30 min, washed, and fixed using 1.5% PFA or eBioscience FoxP3 fixation reagent. Colon lamina propria was obtained as previously described (28 (link)). Cells with the light scatter properties of singlet lymphocytes were analyzed by multicolor immunofluorescence staining and a BD FACS Fortessa II flow cytometer (Becton Dickinson, San Jose, CA). Fcγ receptors blockade was performed (2.4G2; BD PharMingen) prior to surface staining of cell surface markers. The anti-mouse mAbs used in this study included CD4-PE.Cy7/FITC (GK1.5), CD45.1-AF700/Pacific Blue (A20), CD45RB-AF647 (C363-16A) from BioLegend, RORγt-PE (Q31-378) from BD PharMingen, FoxP3-APC (FJK-16s) from eBioscience, and CD45.2-PE (104) from Tonbo. The LIVE/DEAD® fixable cell death stain kit (Invitrogen) was used to remove dead cells from all analysis and avoid background staining noise of dead cells. All flow cytometry analysis and data display were performed using FlowJo software.

Maseda D., Banerjee A., Johnson E.M., Washington M.K., Kim H., Lau K.S, & Crofford L.J. (2018). mPGES-1-Mediated Production of PGE2 and EP4 Receptor Sensing Regulate T Cell Colonic Inflammation. Frontiers in Immunology, 9, 2954.

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Multiparameter Flow Cytometry for T Cell Subsets

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For analysis of T cell subsets, cells were stained with CD3-PerCp-Cy5 (17A2; BioLegend, San Diego, CA), CD4-APC-Cy7 (RM4-5; BioLegend, San Diego, CA), neuropilin-1-PE (761705; R&D Systems, Minneapolis, MN), helios-FITC (22F6; BioLegend, San Diego, CA), CD25-Bio (7D4), CD25-PE-Cy7 (PC61; BioLegend, San Diego, CA) and FoxP3-APC (FJK-16s; eBioscience, San Diego, CA). Intracellular staining was performed with a FoxP3 staining kit (eBioscience), as described previously ). Flow cytometry was performed on a BD FACSCanto II system (BD Biosciences, San Jose, CA), and data were analyzed by FlowJo (10.0.8) software (FlowJo LLC, Ashland, OR).

Schwarz C., Unger L., Mahr B., Aumayr K., Regele H., Farkas A.M., Hock K., Pilat N, & Wekerle T. (2016). The Immunosuppressive Effect of CTLA4 Immunoglobulin Is Dependent on Regulatory T Cells at Low But Not High Doses. American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons, 16(12).

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T Cell Flow Cytometry Panel Protocol

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The T cell flow cytometry panel included the following directly conjugated primary antibodies: anti-mouse CD45 superbright 600 clone: 30-511 (ref# 63-0451-82, eBioscience), anti-CD3 APC-Cy7 clone 17A2(BD Biosciences, cat # 560590), eBioscience anti-mouse CD4 PE-Cy7 clone: RM4-5 (Ref # 25-0042-82, Invitrogen), PE anti-mouse CD8a (Ly-2)(53-6.7) (cat # 553032, BD), anti-mouse CD69 FITC clone: H1.2F3 (Ref# 11-0691-81, eBioscience), and Foxp3 (FJK-16s) APC (eBioscience). Gating strategies are as follows:
CD4+ T cell: live/CD45+/CD3+/CD4+/Foxp3-
CD8+ T cell: live/CD45+/CD3+/CD8+
Treg: live/CD45+/CD3+/CD4+/Foxp3+
Activated CD8+ T cell: live/CD45+/CD3+/CD8+/CD69+

Huntington K.E., Louie A.D., Srinivasan P.R., Schorl C., Lu S., Silverberg D., Newhouse D., Wu Z., Zhou L., Borden B.A., Giles F.J., Dooner M., Carneiro B.A, & El-Deiry W.S. (2023). GSK-3 Inhibitor Elraglusib Enhances Tumor-Infiltrating Immune Cell Activation in Tumor Biopsies and Synergizes with Anti-PD-L1 in a Murine Model of Colorectal Cancer. International Journal of Molecular Sciences, 24(13), 10870.

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