For suppression assays, cells were washed with 0.1% (w/v) sodium azide/phosphate-buffered saline (Mediatech Cellgro) on day 7 of in vitro stimulation and were stained with anti-CD4 PECy5 (BD Pharmingen), anti-CD8 Pacific Blue (Biolegend), and anti-CD25 APCCy7 (BD Pharmingen. For surface phenotyping of cells, bulk PBMCs and enriched CD8+ T cells were stained with anti-CD3 Alexa 700 (BD Pharmingen), anti-CD8 AmCyan (BD Biosciences), anti-CD27 APCCy7 (Biolegend), anti-CD28 APC (BD Pharmingen), CD45RO Pacific Blue (Biolegend), anti-CD62L PECy5 (BD Pharmingen), and anti-CD57 PE (Southern Biotech). For intracellular staining of cytokines, cells were initially activated with 1 μL of leukocyte activation cocktail (BD Pharmingen) for 5 hours. Cells were surface stained with anti-CD8 APC (BD Biosciences), anti-CD4 PECy5 (BD Pharmingen) and anti-CD25 APCCy7 (BD Pharmingen) and permeabilized as described previously. Intracellular staining was performed using anti-IFNγ PECy7 (BD Pharmingen), anti-IL-17A PE (Ebioscience), anti-Granzyme B Alexa 700 (BD Pharmingen) and anti-Perforin Pacific Blue (BD Pharmingen). All cells were resuspended in 1% paraformaldehyde (Electron Microscopy Sciences, Hatfiled, PA) for FACS analysis. Flow cytometric data were acquired on a 4-Laser, 17-color LSRII using FACSDiva software (Becton Dickinson). CFSE was detected in the FITC channel on the LSR.
Anti cd25 apc cy7
Manufactured by BD
Sourced in United States
Anti-CD25 APC-Cy7 is a fluorescent-conjugated antibody that binds to the CD25 antigen. CD25 is a cell surface marker expressed on activated T cells, regulatory T cells, and other immune cell subsets. This product can be used in flow cytometry applications to identify and quantify CD25-positive cell populations.
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Lab products found in correlation
Anti cd3 alexa 700, by BD (2 mentions)
Live dead fixable aqua dead cell stain kit, by Thermo Fisher Scientific (1 mentions)
Anti cd8 percp, by BD (1 mentions)
Anti cd44 pe, by BD (1 mentions)
Pe cy7 anti ifnγ, by BD (1 mentions)
Anti cd86 apc, by BD (1 mentions)
Fitc anti il17a, by BioLegend (1 mentions)
Pacblue anti cd4, by BD (1 mentions)
Anti il 10 pe, by Thermo Fisher Scientific (1 mentions)
Brefeldin a, by Merck Group (1 mentions)
Anti cd4 fitc, by BD (1 mentions)
Saponin, by Merck Group (1 mentions)
Paraformaldehyde (pfa), by Merck Group (1 mentions)
Ionomycin, by Merck Group (1 mentions)
Phorbol myristate acetate (pma), by Merck Group (1 mentions)
Macsquant analyzer, by Miltenyi Biotec (1 mentions)
Anti cd62l apc, by BD (1 mentions)
Anti cd4 pe, by BD (1 mentions)
Apc anti human cd127, by BioLegend (1 mentions)
Anti pd 1 pe, by BioLegend (1 mentions)
8 protocols using anti cd25 apc cy7
1
Phenotypic and Functional Profiling of T Cells
2
Multiparametric Flow Cytometry for Immune Cell Profiling
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Surface staining was performed using following antibodies: FITC-anti-CD4 (Clone RM4-5; 1:200), PerCP-anti-CD8 (Clone 53-6.7; 1:100), PacBlue-anti-B220 (Clone RA3-6B2, 1:200), APC-Cy7-anti-CD25 (Clone PC61, 1:200), PE-anti-CD44 (Clone IM7, 1:200), and APC-anti-CD86 (Clone B7-2, 1:200) (all from BD Biosciences, San Jose, CA, USA).
For intracellular staining, cells were restimulated for 5 h with 10 ng/ml phorbol myristate acetate and 1 µg/ml ionomycin, in a tissue culture incubator at 37°C (both Sigma-Aldrich). Ten micrograms per milliliter brefeldin A (Sigma-Aldrich) were added to the cell suspensions after 1 h of polyclonal restimulation. Then cells were treated with LIVE/DEAD Fixable Aqua Dead Cell Stain kit (Life Technologies) and hereafter fixed with 2% paraformaldehyde (Sigma-Aldrich) for 20 min at room temperature. Cells were stained in 0.5% saponin (Sigma-Aldrich) using following antibodies: PacBlue-anti-CD4 (Clone RM4-5; 1:400), PE-Cy7-anti-IFN-γ (Clone XMG 1.2; 1:400) (both from BD Biosciences), FITC-anti-IL17A (Clone TC11-18H10.1; 1:200, BioLegend, San Diego, CA, USA), PE-anti-IL10 (Clone JESS-16E3; 1:100), and APC-anti-IL22 (Clone IL22JOP; 1:100) (both from eBioscience). All data were acquired on a MACSQuant analyzer (Miltenyi Biotec, Bergisch Gladbach, Germany) and were analyzed with FlowJo Software v10.1 (Tree star, Ashland, OR, USA).
For intracellular staining, cells were restimulated for 5 h with 10 ng/ml phorbol myristate acetate and 1 µg/ml ionomycin, in a tissue culture incubator at 37°C (both Sigma-Aldrich). Ten micrograms per milliliter brefeldin A (Sigma-Aldrich) were added to the cell suspensions after 1 h of polyclonal restimulation. Then cells were treated with LIVE/DEAD Fixable Aqua Dead Cell Stain kit (Life Technologies) and hereafter fixed with 2% paraformaldehyde (Sigma-Aldrich) for 20 min at room temperature. Cells were stained in 0.5% saponin (Sigma-Aldrich) using following antibodies: PacBlue-anti-CD4 (Clone RM4-5; 1:400), PE-Cy7-anti-IFN-γ (Clone XMG 1.2; 1:400) (both from BD Biosciences), FITC-anti-IL17A (Clone TC11-18H10.1; 1:200, BioLegend, San Diego, CA, USA), PE-anti-IL10 (Clone JESS-16E3; 1:100), and APC-anti-IL22 (Clone IL22JOP; 1:100) (both from eBioscience). All data were acquired on a MACSQuant analyzer (Miltenyi Biotec, Bergisch Gladbach, Germany) and were analyzed with FlowJo Software v10.1 (Tree star, Ashland, OR, USA).
3
Phenotyping of T/CAR-T cells
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T/CAR-T cells were centrifuged at 300×g for 5 minutes and resuspended in 100 μL PBS with 2% FBS. The T/CAR-T cells were stained at 4°C for 30 minutes with the following surface marker-specific antibodies: APC/Cy7-anti-CD25 (BD Pharmingen, San Diego, CA, USA), APC-anti-CD69 (BioLegend, San Diego, CA, USA), PE-anti-CD4 (BD Pharmingen), PE/Cy7-anti-CD8 (BioLegend), PE-anti-CD45RO (BD Pharmingen), APC-anti-CD62L (BD Pharmingen), PE-anti-PD-1 (BioLegend), PE/Cy7-anti-CTLA-4 (BioLegend), APC-anti-TIM-3 (BioLegend), and APC anti-human CD127 (BioLegend). The cells were then washed twice and resuspended in 400 μL PBS with 2% FBS for flow cytometry.
4
CD4+ T Cell Polarization Assay
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Naïve CD4+ T cells were sorted from mouse splenocytes as described above. 200,000 cells were stimulated with 4 µL/well CD3/CD28 Dynalbeads in 96 well U bottom plates in 200 µL XVIVO-20 media in the presence of CA or DMSO vehicle control. TGF-β was added to some wells. On days 2 and 4, 100 µL of media were removed, and 100 µL of 2× cytokine/metabolite were re-added. On day 5, Dynalbeads were removed magnetically and cells were stained for flow analysis. Cells were stained with anti-CD4-QDot 605, anti-CD8-PeCy5.5, Aqua viability dye, anti-CD3-PB, and anti-CD25-APCCy7 (BD). Cells were fixed and permeabilized with the FOXP3/Transcription Factor Staining Buffer Set (eBioscience) and stained intranuclearly with anti-FOXP3-PE (eBioscience).
5
Multiparameter Flow Cytometry Immunoassay
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Antibodies used for surface staining included anti-CD14 Qdot 655, anti-CD20 Qdot 655, anti-CD4 PeCy5.5, anti-CD8 Qdot605 (Invitrogen; Carlsbad, California), anti-CD14 BV650, anti-CD20 BV650 (Biolegend; San Diego, California), anti-CD28 ECD (Beckman Coulter; Fullerton, CA), anti-CD25 APC-Cy7, anti-CD95 PE-Cy5, and anti-HLA-DR APC (BD Pharmingen; San Diego, California). Antibodies used for intracellular staining included anti-interleukin-2 (IL-2) Alexa700 (Biolegend), anti-interferon-gamma (IFNγ) Alexa 700 (Invitrogen), anti-tumor necrosis factor-alpha (TNFα) Alexa 700, anti-CD3 Pac Blue, anti-CD69 PE, and anti-Ki67 FITC (BD Pharmingen).
6
Comprehensive Immune Cell Profiling in Sepsis
7
Multiparameter Flow Cytometry of Immune Cells
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RBCs were lysed using appropriate buffer (BD Pharm Lyse™, #555899, BD Biosciences, Franklin Lakes, NJ). Each sample was adjusted to a concentration of 107 WBCs/mL with cold (4°C) FACS buffer (PBS with 2% BSA), and a 100 μL aliquot was placed into a polystyrene tube. Aliquots of stock antibody solutions (20 μL) of anti‐CD45+ APC/Cyanine7, anti‐CD3+ FITC, anti‐CD4+ PE/Cyanine7, anti‐CD8+ APC, anti‐CD197(CCR7)+ APC/Cyanine7 (BioLegend, San Diego, CA, USA), and anti‐CD14+ PE, anti‐CD69+ PE, anti‐CD45RA+ PE, anti‐CD25+ APC‐Cy™7 (BD Biosciences), were added to each sample, and samples were incubated at 4°C for 30 min in the dark. Next, samples were washed (twice) with 2 mL of FACS buffer by centrifugation at 300 × g for 5 min at 4°C. The supernatant was discarded, and labeled cells were re‐suspended in 100 μL of FACS buffer. A 5 μL aliquot of the viability dye (7AAD, BioLegend) was added to each sample and incubated for 10 min at room temperature in the dark. Samples were analyzed using a flow cytometer (Novocyte 2006R, Ace Bioscience, San Diego, CA, USA) with all necessary controls (unstained and compensation).
8
Multiparametric Flow Cytometry Analysis
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For surface staining, cells were stained with anti-CD3 PerCP-Cy5.5, anti-CD4 APC, anti-CD25 APC-Cy7, anti-CD69 Pe-Cy7, anti-PD-1 BV421, and anti-CD38 Alexa Fluor 488 (all from BD Biosciences, San Diego, CA, USA). Cells were then incubated for 15 minutes under room temperature and were fixed with 1% formaldehyde for 15 minutes. For intracellular staining, cells were permeabilized (Cytofix/Cytoperm TM Fixation/Permeabilization Solution Kit, BD Biosciences) for 40 minutes and stained with anti-FoxP3 Alexa Fluor 488, anti-CTLA-4 BV605, and anti-IL-10 APC. All the samples were analyzed using an LSRFortessa flow cytometer (Becton-Dickinson, San Jose, CA, USA) and analyzed by FlowJo software (FlowJo, Tree Star, Ashland, OR, USA).
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