PerCp-Cy5.5 anti-CD161, PE anti-RORγt, PE anti–γδ TCR, biotinylated anti–γδ TCR, PerCp-Cy5.5 anti-CD45RA, and APC anti–αβ TCR antibodies were from eBioscience. A647 anti-PLZF, APC-Cy7 anti-CD4, PE-Cy7 anti-CD8, BV421 anti-CD3, BV421 anti-CD161, PE anti-STAT4 (pY693), PE anti–IL-6R, PE anti–IL-12Rβ1, PE anti–IL-21R, PE mouse IgG1 isotype control, PerCP-Cy5.5 anti-STAT3 (pY705), and FITC anti–Vδ2 TCR antibodies were from BD. FITC anti–Vα24 TCR and PE and biotinylated anti-Vβ11 antibodies were from Beckman Coulter. FITC anti–Vδ1 TCR was from Thermo Fisher Scientific. APC or APC-Cy7 anti-Vα7.2 and PE-Cy7 anti–αβ TCR antibodies were from BioLegend. FITC anti-CCR7 antibody was from R&D Systems. MR1–5-OP-RU tetramers have been described previously (Reantragoon et al., 2013 (link); Corbett et al., 2014 (link)).
Anti cd4 apc cy7
Manufactured by BD
Sourced in United States
Anti-CD4 APC-Cy7 is a fluorescently-labeled monoclonal antibody that specifically binds to the CD4 cell surface marker. It is designed for use in flow cytometry applications to identify and quantify CD4-positive cells within a sample.
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Lab products found in correlation
Anti cd25 pe cy7, by BD (6 mentions)
Anti cd3 fitc, by BD (5 mentions)
Anti cd27 pe, by BD (4 mentions)
Anti cd3 pe cy7, by BD (4 mentions)
Facscanto 2 flow cytometer, by BD (4 mentions)
Cytofix cytoperm, by BD (4 mentions)
Anti ifn γ apc, by BD (4 mentions)
Facscanto 2, by BD (4 mentions)
Anti cd8 pe cy7, by BD (3 mentions)
Anti cd3 bv421, by BD (3 mentions)
Anti cd8 percp, by BD (3 mentions)
Anti cd8 apc, by BD (3 mentions)
Anti cd127 bv650, by BioLegend (3 mentions)
Anti il 2 bv711, by BD (3 mentions)
Anti il 17f bv786, by BD (3 mentions)
Anti tnf α buv395, by BD (3 mentions)
Anti il 13 bv421, by BD (3 mentions)
Anti il 17a apccy7, by BioLegend (3 mentions)
Fitc anti foxp3, by BD (2 mentions)
Cytofix cytoperm fixation and permeabilization kit, by BD (2 mentions)
49 protocols using anti cd4 apc cy7
1
Multiparameter Immunophenotyping of Immune Cells
2
Comprehensive Immune Profiling of Tumor Microenvironment
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Flow cytometry was performed on venous peripheral blood collected in heparin-coated vacutainer tubes, on tumor and peritumoral tissues using a FACSCanto II 6-colour flow cytometer, daily calibrated with calibrite beads (Fitc, Pe, PerCP and APC) and compbeads (Pe-Cy7 and APC-Cy7; Becton Dickinson, San Jose, CA, USA). Fluorochrome-labelled monoclonal antibodies (BD Bioscience) for identification of circulating and tissue Treg cells were used: Fitc-anti-FOXP3, Pe-Cy7-anti-CD25, APC-Cy7-anti-CD4, APC-anti-CD45RA, Pe-anti-CD152 (CTLA-4), PercP-anti-CD184 (CXCR4), APC-anti-CD279 (PD-1), Pe-anti-CD278 (ICOS) and APC-anti-CD39 (ENTPD1). Intracellular staining for FoxP3, ICOS and CTLA-4 was performed using a commercially available kit (BD Cytofix/Cytoperm; fixation and permeabilization kit; BD Pharmingen) according to the manufacturer's instructions. A minimum of 100.000 events for each sample were collected and data were analysed using FacsDiva software 6.1.3 (BD Bioscience). The absolute number of CD4 was calculated as follow: [total withe blood cell count (cells/uL) x percent CD4]/100 or [total tumor-infiltrating immune cell count (cells/100 mg tumor) x percent CD4]/100.
3
Profiling Immune Cell Populations in Murine Tissues
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Lymphocytes were isolated from the peripheral blood, spleen, bone marrow and lymph nodes of the indicated mice. Total cell numbers were counted using counting slides (SD-100, Nexcelom) in Cellometer Mini Automated Cell Counter (Nexcelom). Surface antigens were stained with indicated conjugated primary antibodies in the staining buffer (1 × PBS, 3% BSA, 1 mM EDTA, 0.1%NaN3) at 4 °C for 30 min. Antibodies used are asfollows: FITC anti-CD3 (11-0031-82, eBioscience), APC Cy7 anti-CD4 (552051, BD Biosciences), PerCp anti-CD8 (100732, Biolegend), PE anti-B220 (12-0452-83, eBioscience), APC anti-B220 (17-0452-83, eBioscience), APC anti-CD11b (17-0112-83, eBioscience), Brilliant Violet 421 anti-CD11b (562605, BD Biosciences), PE Cy7 anti-CD19 (25-0193-82, eBioscience), FITC anti-IgM (115-097-020, Jackson Laboratories), FITC anti-F4/80 (11-4801-85, eBioscience) were used for flow cytometry analysis in this study. After staining, cells were washed once with 1XPBS and immediately analyzed by in cytoflex S flow cytometer (cytoflex S, Beckman Coulter). All analyses were performed using CytExpert software (CytExpert, Beckman Coulter, Inc.).
4
Analyzing B cell subtypes in T-B cell co-culture
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WBCs were isolated as described above and analyzed with a FACS Aria flow cytometer (Beckton Dickinson) to obtain CD4+CXCR5− T cells, CD4+CXCR5+ cells, and CD19+CD27+ memory B cells (mB) (4×105/mL of each cell type). The following fluorescently labeled antibodies were used for fluorescence-activated cell sorting (FACS) analysis: APC anti-CXCR5, APCCy7 anti-CD4, PE anti-CD27, PECy7 anti-CD19, and BV421 anti-CD38 (all from BD Bioscience).
Cells were maintained in RPMI 1640 medium supplemented with 50% fetal bovine serum (FBS). For co-culture experiments, 1×105 T and B cells were added to wells of a 96-well plate and co-cultured in 200 µL complete medium containing 10% FBS and 0.2 µg/mL staphylococcal enterotoxin B (SEB). Half of the medium was changed every 2 days to keep the SEB concentration constant. After 6 days of culture, co-cultured cells were subjected to flow cytometry, based on positive staining against the markers fixable viability dye (FVD), CD4, CXCR5, CD19, CD27, and CD38 to reveal changes in the B cell subtype.
Cells were maintained in RPMI 1640 medium supplemented with 50% fetal bovine serum (FBS). For co-culture experiments, 1×105 T and B cells were added to wells of a 96-well plate and co-cultured in 200 µL complete medium containing 10% FBS and 0.2 µg/mL staphylococcal enterotoxin B (SEB). Half of the medium was changed every 2 days to keep the SEB concentration constant. After 6 days of culture, co-cultured cells were subjected to flow cytometry, based on positive staining against the markers fixable viability dye (FVD), CD4, CXCR5, CD19, CD27, and CD38 to reveal changes in the B cell subtype.
5
CD4+CD25+ T Cell Activation Assay
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Isolated CD4+CD25+ T cells were incubated for 30’ at 37°C in 5% CO2 with or without 10 μM of Peptide R29. After 30’ the cells were washed and then stained with Fitc-anti-CD25, APC-Cy7-anti-CD4 (BD Bioscience) and 7-amino-actinomycin D (7-AAD) (BioLegend) and analyzed by flow cytometer.
6
DSS and T-cell Transfer Colitis Models
7
Multiparameter Flow Cytometry Analysis
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FITC-conjugated major histocompatibility complex (MHC) class I–specific antibody (IgG2a, W6/32, CBL139F, Cymbus Biotech, Hants, UK) and PE anti-human CD155/PVR (clone SKIL.4, Biolegend, Cat No 337609) were used for flow cytometry. FACSAria II Cell Sorter 8-colour flow cytometer with BD FACSDiva™ 8.0 Software (BD Biosciences, San Jose, CA) was daily calibrated with calibrite beads (Fitc, Pe, PerCP and APC) and compbeads (Pe-Cy7 and APC-Cy7; BD Biosciences). PerCP-Cy5.5 anti-CD3 (BD Biosciences Cat No 560835), FITC-conjugated anti-CD56 (BD Biosciences, Cat No 345811) and PE-conjugate anti-CD107a antibody (BD Bioscience, Cat No 555801) were used to identify degranulating NK-cells from peripheral blood. For intracellular staining PE-conjugated anti-IFN-γ antibody (BD Biosciences Cat No 554701) was used. Fluorochrome-labelled monoclonal antibodies for identification of tissue Treg cells were: FITC-anti-FOXP3 (BD Biosciences, Cat No 560047), Pe-Cy7-anti-CD25 (BD Biosciences, Cat No 557741) and APC-Cy7-anti-CD4 (BD Biosciences, Cat No 557871). Intracellular staining for FoxP3 was performed using a commercially available kit (BD Cytofix/Cytoperm; fixation and permeabilization kit; BD Pharmingen) according to the manufacturer’s instructions. A minimum of 100.000 events for each sample were collected.
8
T Cell Immunophenotyping and Viability
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PBMCs or T cells were stained with PE-Cy7 anti-CD3 (BD, 560910), APC-Cy7 anti-CD4 (BD, 557871), BV510 anti-CD8 (BD, 344732) for T cell subset analysis. Propidium iodide (PI, 556547, BD Bioscience) were used for cell death analysis according to manufacturer’s instruction. Data were acquired using the flow cytometer (BD verse) and analyzed with the Flowjo software.
9
Multi-Dimensional Immune Cell Profiling
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Single-cell suspensions were stained for 30 min using the following fluorophore-conjugated antibodies: FITC anti-CD3 (BD Biosciences Cat# 555332, RRID:AB_395739), APC/Cy7 anti-CD4 (BD Biosciences Cat# 341095, RRID:AB_400218), Percp-Cy5.5 anti-CXCR5 (BD Biosciences Cat# 562781, RRID:AB_2313576), PE anti-PD-1 (BD Biosciences Cat# 560795, RRID:AB_2033989), PE/Cy7 anti-CCR6 (BD Biosciences Cat# 560620, RRID:AB_1727440), APC anti-CCR6 (BD Biosciences Cat# 560619, RRID:AB_1727439), PECy7 anti-CXCR3 (BD Biosciences Cat# 560831, RRID:AB_2033944), APC anti-CXCR3 (BD Biosciences Cat# 565223, RRID:AB_2687488), FITC anti-CD19 (BD Biosciences Cat# 555412, RRID:AB_395812), BV421 anti-CD27 (BioLegend Cat# 302823, RRID:AB_10900425), APC anti-CD38 (BD Biosciences Cat# 555462, RRID:AB_398599), PECy7 anti-IgD (BD Biosciences Cat# 555776, RRID:AB_396111), and PE anti-CD138 (BD Biosciences Cat# 552026, RRID:AB_394323). For intracellular staining, cells were permeabilized using the Foxp3 Staining Buffer Set (eBioscience), then stained with PECy7 anti-Ki67 (BD Biosciences Cat# 561283, Clone B56), PE anti-Foxp3 (BD Biosciences Cat# 560082, RRID:AB_1645509), and PE anti-Bcl2 (BD Biosciences Cat# 340576, RRID:AB_400061). All samples were analyzed with an LSRFortessa flow cytometer (Beckton Dickinson), and data were processed with FlowJo software, version 10 (TreeStar, Inc.).
10
Multi-Parameter Flow Cytometry and Immunohistochemistry
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Anti‐human mAbs including APC‐anti‐CD3 (UCHT1), FITC‐anti‐CD3 (UCHT1), APC‐Cy7‐anti‐CD4 (RPA‐T4), PE‐anti‐CD8 (SK1), APC‐anti‐CD8 (SK1), PE‐Cy7‐anti‐CD38 (HIT2), PE‐anti‐PD‐1 (EH12.1), PerCP‐Cy5.5‐anti‐CXCR5 (RF8B2), PE‐anti‐CD27 (M‐T271), FITC‐anti‐CD27 (M‐T271), FITC‐anti‐CD45RA (HI100), APC‐anti‐CCR6 (11A9), PE‐Cy7‐anti‐CXCR3 (1C6), and APC‐Cy7‐anti‐CD19 (SJ25C1) (BD Biosciences) and anti‐mouse mAbs including PE‐Cy7‐anti‐CD3 (145‐2C11), PerCP‐Cy5.5‐anti‐CD4 (RM4‐5), PE‐anti‐B220 (RA3‐682), PE‐anti‐Bcl‐6 (K112‐91), PE‐anti‐T‐bet (4B10), PE‐anti‐GATA3 (L50‐823), PE‐anti‐RORγt (Q31‐378) (BD Bioscience), APC‐anti‐PD‐1 (29.F1A12), FITC‐anti‐CXCR5 (L138D7), and Pacific Blue‐anti‐CD45.2 (104) (Biolegend) were used for flow cytometry. For immunohistochemistry, we used anti‐human Abs including Alexa Fluor 647‐anti‐CD4 (MT310), mouse anti‐Bcl6 (P1F6; Nichirei, Tokyo, Japan), and rabbit anti‐Bob1 pAb (C‐20; Santa Cruz Biotechnology) for staining of human tonsils and we used hamster anti‐mouse CD3 (500A2; Life Technologies) and biotin‐PNA visualized by streptavidin‐PE (Vector Laboratory) for staining of mouse spleens.
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