Fluorochrome conjugated antibodies CD3-BV421 (HIT3a; 740073), CD25-BV786 (M-A251; 563701), PD-1-BV711 (EH12.1; 564017), PD-L1-BV605 (MIH1; 740426), CD38-BV786 (HIT2; 563964), CD14-FITC (M5E2; 557153), CD19-BV711 (SJ25C1; 563036), ROR𝛾t-AF488 (Q21-559; 563621) and Bcl-2-BV450 (Bcl-2/100; 560637) were from BD Biosciences (PA, US). CD4-AF700 (OKT-4; 56-0048-82), FOXP3-PE-Cyanine7 (236A/E7; 25-4777-42), CD3-FITC (HIT3a; 11-039-42), IL-1β-PE (CRM56; 12-7018-82), and IFN-γ-APC (4S-B3; 17-7319-82) were from Thermo Fisher Scientific (CA, US). T-bet- PerCP-Cyanine5.5 (eBio4B10; 45-5825-82) and LAG3- PerCP-eFluor™ 710 (3DS223H; 46-2239-42) are from eBiosciences (CA, US). NLRP3-PE (bs-10021R-PE) is from Bioss (MN, US). TCR stimulating antibodies used in this study were for activating CD3 (HIT3a; 555336;BD biosciences; PA, US) and CD28 (CD28.2; 16-0289-85; Life Technologies corporation; CA, US). Recombinant human IL-1β (C600124-0010) and IL-2 cytokines were from BioBasic Inc. (NY, US). Anakinra (Kineret from Amgen) was a kind gift from Dr. Su at NIAID, NIH.
Cd25 bv786
Manufactured by BD
Sourced in United States
The CD25-BV786 is a fluorochrome-conjugated monoclonal antibody designed for flow cytometric analysis of the CD25 (IL-2Rα) antigen expressed on activated T cells and regulatory T cells.
Automatically generated - may contain errors
Lab products found in correlation
Cd14 fitc, by BD (1 mentions)
Cd19 bv711, by BD (1 mentions)
Cd3 bv421, by BD (1 mentions)
Pd 1 bv711, by BD (1 mentions)
Cd38 bv786, by BD (1 mentions)
Cd3 fitc, by Thermo Fisher Scientific (1 mentions)
Pd 1 apc, by BD (1 mentions)
Cd4 bv605, by BD (1 mentions)
Tigit bv421, by BD (1 mentions)
Lag3 bv711, by BD (1 mentions)
Carboxyfluorescein succinimidyl ester (cfse), by Thermo Fisher Scientific (1 mentions)
Cd8 apc h7, by BD (1 mentions)
Cd4 pe, by Beckman Coulter (1 mentions)
Cd45ra fitc, by BD (1 mentions)
Cxcr3 bv711, by BD (1 mentions)
Ccr6 percp cy5, by BD (1 mentions)
Cd127 buv395, by BD (1 mentions)
Ficoll density centrifugation, by Merck Group (1 mentions)
Ccr7 pe, by BD (1 mentions)
Facscelesta, by BD (1 mentions)
4 protocols using cd25 bv786
1
Comprehensive T-cell Phenotyping and Cytokine Analysis
2
Multiparametric Flow Cytometry Analysis
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Cell phenotypes were evaluated by multiparameter flow cytometry. Cells were incubated with a panel of labeled monoclonal antibodies (mAbs) anti-CD3 AF700, CD4 BV605, CD8 APC-H7, CD25 BV786, PD-1 APC, TIGIT BV421, and LAG3 BV711 (BD Biosciences) in staining buffer on ice in the dark for 20 min. Cells were then washed in the staining buffer and re-suspended in staining buffer and analyzed on an LSRII flow cytometer. Isotype controls were used for each experiment. Analysis of the results used FlowJo 10 software.
CFSE staining was conducted according to manufacturer’s instructions (Thermo Fisher Scientific). Briefly, cells were labeled with CFSE by adding 1 mL of freshly prepared CFSE (2 μM in PBS containing 2% EV free FCS) to cells (up to 1×108 cells) in 1 mL of PBS 2% EV free FCS. The tube containing this mixture was covered with foil and incubated at 337°Cfor 5 minutes. Cells were pelleted, washed twice with 10 mL of PBS 2% EV free FCS, resuspended, counted and used for experiments.
CFSE staining was conducted according to manufacturer’s instructions (Thermo Fisher Scientific). Briefly, cells were labeled with CFSE by adding 1 mL of freshly prepared CFSE (2 μM in PBS containing 2% EV free FCS) to cells (up to 1×108 cells) in 1 mL of PBS 2% EV free FCS. The tube containing this mixture was covered with foil and incubated at 337°Cfor 5 minutes. Cells were pelleted, washed twice with 10 mL of PBS 2% EV free FCS, resuspended, counted and used for experiments.
3
Multiparametric Characterization of CD4+ T Cells
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Peripheral blood mononuclear cells (PBMCs) were obtained after Ficoll density centrifugation (Sigma-Aldrich, Burlington, MA). CD4+ T cells were purified from blood using negative selection on magnetic columns (CD4 T-cell isolation kit, human; Miltenyi Biotec, Bergisch Gladbach, Germany). Cells were stained using the following antihuman antibodies: CXCR5-BV605, CD45RA-FITC, CCR7-PE, CXCR3-BV711, CCR6- PercpCy5.5, PD-1-BUV737, CD25-BV786, and CD127-BUV395 (BD Biosciences, Franklin Lakes, NJ). For CSF samples and transmigration assays, CCR7-PE was replaced by a CD4-PE (Beckman Coulter, Brea, CA). Cells were analyzed by flow cytometry (FACS Celesta, BD Biosciences). Dead cells were excluded from the analysis using live/dead Amcyan staining (Invitrogen, Waltham, MA).
4
Treg Secretion Assay for Immune Profiling
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For the T-regulatory cell (Treg) secretion assay, peripheral blood mononuclear cells were thawed and rested 1 night in complete medium (RPMI-160, 10% fetal bovine serum, 1% glutamine, 1% penicillin/streptomycin; 5 million/ml). PBMCs were then cultured at 5 million/ml in complete medium and activated for 5 hours with phorbol 12-myritate 13-acetate (PMA, 50 ng/ml), ionomycin (1 µg/ml), and brefeldin A (1×) (Sigma Aldrich, St. Louis, MO) to block intracellular trafficking. The following antibodies and reagents were used for flow cytometry: CD3-BUV395 (UCHT1); CD25-BV786 (M-A251); CD4-BV650 (SK3); CD127-BV450 (HIL-7R-M21); IL-10-APC (JES3-19F1); IL-17A-PE (SCPL1362); IL-13-PE (JES10-5A2); IL-4-APC (MP4-25D2); and IfnG-FITC (B27) (BD Biosciences, Heidelberg, Germany); CD45RA-APCeFluor780 (HI-100); and FoxP3-PercpCy5.5
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