Foxp3 pe fjk 16s

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The Foxp3 PE (FJK-16s) is a flow cytometry reagent used for the detection and analysis of Foxp3-expressing regulatory T cells. It consists of a phycoerythrin (PE)-conjugated monoclonal antibody specific for the Foxp3 transcription factor, which is a key marker of regulatory T cells.

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6 protocols using foxp3 pe fjk 16s

Identification of FoxP3+ Regulatory T Cells

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Cells expressing FoxP3 were identified among spleen and lymph node cells, as well as cells from peripheral blood, using the FoxP3 staining kit provided by eBioscience and following the manufacturer’s protocol. Cells were incubated with Fc block (clone 2.4G2, BioXcell) and stained with the following antibodies: eBioscience anti-CD45-FITC (30-f11), anti-CD4-PerCP ef710 (RM4.5) and FoxP3-PE (FJK-16s) were purchased from Invitrogen (Carlsbad, CA). Anti-CD25-APC (PC61.5) and anti-CD8-PE-Cy7 were purchased from Biolegend (San Diego, CA). For FoxP3 staining, eBioscience FoxP3/transcription factor staining buffer set (Cat. Nr 00-5523-00) (Invitrogen) was used according to the manufacturer’s protocol. Tonbo Biosciences Ghost Dye™ Red 780 (Cat No. 13-0865) was used for excluding the dead cells. The staining patterns were processed by CytoFLEX (Beckman Coulter, Brea, CA) and data were analyzed by FlowJo v. 10 (Tree Star, Ashland, OR, USA).

Xu B., Tang J., Lyu C., Wandu W.S., Stumpo D.J., Mattapallil M.J., Horai R., Gery I., Blackshear P.J, & Caspi R.R. (2021). Regulated Tristetraprolin Overexpression Dampens the Development and Pathogenesis of Experimental Autoimmune Uveitis. Frontiers in Immunology, 11, 583510.

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Multiparameter Immunophenotyping of Lymphocytes

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Single-cell leukocyte suspensions from the spleen and draining lymph nodes were generated by gentle dissection. The following mAbs were used for staining: anti CD4-PE (L3T4 GK1.5), and anti-B220-Fluorescein isothiocyanate (FITC; RA3-6B2) from BD Biosciences; anti CD8a-Pacific Blue (53–6.7) from eBioscience (Waltham, MA, USA); anti-CD1d-FITC (1B1), anti-CD5-PE (53–7.3), anti-CD11b-PerCP/Cy5.5 (M1/70), anti-CD19-APC (6D5), anti-CD19-PE-Cy7 (6D5), anti-CD90.2-PE/Cy7 (30-H12), anti-F4/80-APC/Cy7 (BM8), anti-IL-10-PE (JES5-16E3), anti-IL-10-APC (JES5-16E3), and anti-IL-17A-APC (TC11-18H10.1) from BioLegend; and FoxP3-PE (FJK-16 s) from Invitrogen (Carlsbad, CA, USA). LIVE/DEAD Fixable Aqua Dead Cell Stain (Invitrogen) was used to detect dead cells. Single-cell suspensions (10⁶ cells) from the spleen and draining lymph nodes were stained at 4 °C for 20 min using mAbs at predetermined optimal concentrations.

Mizumaki K., Horii M., Kano M., Komuro A, & Matsushita T. (2021). Suppression of IL-23-mediated psoriasis-like inflammation by regulatory B cells. Scientific Reports, 11, 2106.

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Immunophenotyping of Tumor-Infiltrating Cells

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SM1 tumors harvested from mice were digested with collagenase (Sigma-Aldrich). Splenocytes and cells obtained from digested SM1 tumors, were stained with antibodies to CD3 BV605 (clone 17A2), Ly6C FITC (Clone AL-21), PD-L1/CD274 PE (Clone MIH5) (Becton Dickinson Biosciences), CD8a BV421 (Clone 53-6.7) (Biolegend), Ly-6G (Gr1) PerCP 5.5 (clone RB6-8C5), CD11b APC (clone M1/70), F4/80 Pacific blue/eFluor450 (clone BM8), CD25 APC (PC61.5), CD4 FITC (RM4-5) (eBioscience), and analyzed with LSR-II or FACSCalibur flow cytometers (Becton Dickinson Biosciences), followed by analysis using Flow-Jo software (FLOWJO, LLC) as previously described (30 (link)). Intracellular staining of interferon gamma was done as previously described (30 (link)). Intracellular staining of Foxp3 PE (FJK-16s) (eBioscience) was done according to manufacture's recommendations.

Hu-Lieskovan S., Mok S., Moreno B.H., Tsoi J., Faja L.R., Goedert L., Pinheiro E.M., Koya R.C., Graeber T., Comin-Anduix B, & Ribas A. (2015). Improved antitumor activity of immunotherapy with BRAF and MEK inhibitors in BRAFV600E melanoma. Science translational medicine, 7(279), 279ra41.

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Characterization of Tumor-Infiltrating Immune Cells

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SM1 tumors and spleens were harvested from mice. Tumors were further digested with collagenase (Sigma-Aldrich). Splenocytes and cells obtained from digested SM1 tumors, were stained with Ab to CD3 BV605 (clone 17A2), Ly6C FITC (Clone AL-21), PD-L1/CD274 PE (Clone MIH5) (Becton Dickinson Biosciences, San Jose, CA), CD8a BV421 (Clone 53-6.7) (Biolegend, San Diego, CA), Ly-6G (Gr1) PerCP 5.5 (clone RB6-8C5), CD11b APC (clone M1/70), F4/80 Pacific blue/eFluor450 (clone BM8), CD25 APC (PC61.5), CD4⁺ FITC (RM4-5) (eBioscience, San Diego, CA), and analyzed with LSR-II or FACSCalibur flow cytometers (Becton Dickinson Biosciences), followed by analysis using Flow-Jo software (FLOWJO, LLC, Ashland, OR). Intracellular staining of Foxp3 PE (FJK-16s) (eBioscience) was done according to manufacturer's recommendations. After applying a gating strategy for the selection of the target population and exclusion of dead cells in tumors and spleens (Fig. S1A), the different immune cell populations were analyzed. Cells were analyzed with a LSR-II or FACSCalibur flow cytometers (BD Biosciences), followed by Flow-Jo software (Tree-Star, Ashland, OR) analysis as previously described.^{49 (link)}

Homet Moreno B., Mok S., Comin-Anduix B., Hu-Lieskovan S, & Ribas A. (2015). Combined treatment with dabrafenib and trametinib with immune-stimulating antibodies for BRAF mutant melanoma. Oncoimmunology, 5(7), e1052212.

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Multiparametric Flow Cytometry Analysis

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Non-viable cells were excluded from all analyses using Live/Dead eF780 dye (1:1000, eBioscience). Surface staining was performed in PBS containing 0.5% FCS and 2 mM EDTA. Intracellular cytokine staining (ICCS) was performed on cells stimulated with PMA (10 ng/ml) and ionomycin (1 µg/ml) in the presence of GolgiStop (BD Bioscience, 1:1000) for 4 hr. Cells were surface stained before fixation and permeabilization using eBioscience reagents. Staining for intracellular Notch1 and FoxP3 was performed after fixation and permeabilization using FoxP3 staining kit reagents (eBioscience). The following antibodies, all purchased from eBioscience and/or Biolegend, were used; CD4 Alexa700 (GK1.5, 1:100), CD69 FITC (H1.2F3, 1:100), Notch1-PE (mN1A, 1:100, Biolegend), CD8a APC (53–6.7, 1:200), CD19 (1D3, 1:200), B220 FITC (RA3-6B2, 1:100), Vb8.1/2 FITC (KJ16-133, 1:100), FoxP3 PE (FJK-16S, 1:100, eBioscience), CD25 PE-Cy7 (PC61.5, 1:300), IFNγ PE-Cy7 (XMG1, 1:400), IL-2 eF450 (JES6-5H4, 1:100) and IL-17A PE or PE-Cy7 (17B7, 1:2–400).
Soluble IL-17A was detected in culture supernatant by ELISA using Ready-Set-Go ELISA kit (eBioscience).

Britton G.J., Ambler R., Clark D.J., Hill E.V., Tunbridge H.M., McNally K.E., Burton B.R., Butterweck P., Sabatos-Peyton C., Hampton-O’Neil L.A., Verkade P., Wülfing C, & Wraith D.C. (2017). PKCθ links proximal T cell and Notch signaling through localized regulation of the actin cytoskeleton. eLife, 6, e20003.

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IL-17 Intracellular Staining Protocol

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Since IL-17 is di cult to stain in the resting state, so the cells need to be activated rst. Resuspended the cells in RPMI-1640 medium, which contains 10% fetal bovine serum, 100 ng/ml phorbol 12-myristate 13acetate (PMA), 1µg/ml ionomycin and 3µg/ml brefeldin for cells activation [12] . Incubated the cells in 37°C, 5% CO2 incubator for 4 hours. Centrifuged at 500×g for 5 minutes and discarded the supernatant. After blocking with 10% calf serum and incubated at 4°C for 10 minutes, appropriate extracellular antibodies were used for extracellular staining. Then washed the cells, and xed it with xation buffer (Foxp3/Transcription Factor Staining Buffer Set (eBioscience, #00-5523-00)) at 4°C for 30 minutes. The cells were washed with permeabilization buffer and incubated with intracellular antibody (IL17 APC eBio 17B7(eBioscience, #17-7177-81), FOXP3 PE FJK-16s (eBioscience, #12-5773)) at 4°C for 30 minutes. The cells were washed with sterile PBS buffer, and detected with Attune NxT cytometer (Thermo Fisher Scienti c). Image analysis was performed using FlowJo software.

Wen Y., Feng S., Dai H., Mao M., Zhou Z., Li B., Wang C., Cai X., Li S., Yang J., Ren Q, & Sun J. (2022). Intestinal dysbacteriosis-propelled T helper 17 cells activation mediate the perioperative neurocognitive disorder induced by anesthesia/surgery in aged rats. Neuroscience letters, 783.

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