Up to 1.106 cells were labelled in staining solution (PBS, Foetal Calf Serum 2%, 2mM EDTA). Antibodies used were: APC/Cy7 anti-CD3e, PE/Cy7 anti-CD4, APC anti-CD25, PE anti-CD44, FITC anti-CD62L (all from Biolegend). Dead cells were stained either with 7-AAD (Biolegend) or Livedead cell viability assay (Life Technologies). Cells were fixed using fixation solution 20 minutes at room temperature (Biolegend). For regulatory T cells labelling, FOXP3 Perm/Fix solution was use with PE anti-FOXP3 antibody (Biolegend) according to manufacturer’s instructions. Intracellular IFNγ staining was performed using permeabilisation buffer following manufacturer’s instructions and FITC anti-IFNγ antibody and APC anti-IL4 antibody (Biolegend). Data were acquired on a BD FACSCanto II flow cytometer (BD) and analyzed employing FlowJo software (Tree Star).
Anti cd4 pe cy7
Manufactured by BioLegend
Sourced in United States
Anti-CD4-PE/Cy7 is a fluorochrome-conjugated antibody that binds to the CD4 cell surface receptor. It is used for the identification and enumeration of CD4+ T cells in flow cytometry applications.
Automatically generated - may contain errors
Lab products found in correlation
Lsrfortessa flow cytometer, by BD (7 mentions)
Anti cd3 fitc, by BioLegend (7 mentions)
Anti cd8 percp cy5, by BioLegend (6 mentions)
Anti cd8 apc cy7, by BioLegend (5 mentions)
Anti cd25 apc, by BioLegend (4 mentions)
Anti cd8 apc, by BioLegend (4 mentions)
Anti cd3 pe cy7, by BioLegend (4 mentions)
Anti cd4 fitc, by BioLegend (4 mentions)
Anti cd44 pe, by BioLegend (3 mentions)
7 aad, by BioLegend (3 mentions)
Anti tnf α apc, by BioLegend (3 mentions)
Anti cd62l apc cy7, by BioLegend (3 mentions)
Collagenase d, by Merck Group (3 mentions)
Anti cd3 percp cy5, by BioLegend (3 mentions)
Anti cd11b pe, by BioLegend (3 mentions)
Lsrii flow cytometer, by BD (3 mentions)
Dnase, by Merck Group (3 mentions)
Anti cd8 brilliantviolet605, by BioLegend (3 mentions)
Percoll, by GE Healthcare (3 mentions)
Anti cd8 alexa fluor 700, by BioLegend (3 mentions)
52 protocols using anti cd4 pe cy7
1
Multiparametric Flow Cytometry Analysis
2
Immunohistochemical and Flow Cytometry Analysis of Neuroinflammation
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Lipopolysaccharide (Escherichia coli 0111:B4) was obtained from InvivoGen. Recombinant mouse IL4 and IL4I1 were obtained from R&D Systems. The following antibodies were used for immunohistochemistry. Primary antibodies: rat anti-CD11b (1:100; AbD Serotec), rabbit anti-Ym1 (1:100; StemCell Technologies), mouse anti-iNOS (1:50; BD Pharmingen), rabbit anti-Olig2 (1:300; Millipore), mouse anti-CC1 (1:300; Millipore), mouse anti-Nkx2.2 (1:100; DSHB), mouse anti-GFAP (1:400; Sigma), rat anti-Tenascin-C (1:100, Abcam), rabbit anti-NF200 (1:100; Sigma), mouse anti-SMI-32 (1:1000; Calbiochem), mouse anti-IST-9 (1:200; Abcam). Secondary antibodies: Alexa Fluor® 488 Goat Anti-Rabbit IgG (1:1000), Alexa Fluor® 488 Goat Anti-Rat IgG (1:500), Alexa Fluor® 594 Goat Anti-Mouse IgG (1:1000), Alexa Fluor® 594 Chicken Anti-Goat IgG (1:500) and Alexa Fluor® 594 Goat Anti-Rat IgG (1:500). Flow cytometry primary antibodies: PE/Cy7 anti-CD4 (BioLegend), Brilliant Violet 711 anti-T-bet (Biolegend), PE anti-RORγt (BD Pharmingen) and PerCP/Cy5.5 anti-Gata3 (BioLegend), anti-NOS2 PE (Santa Cruz Biotechnology) and anti-CD11b APC/Cy7 (Biolegend). LIVE/DEAD® Fixable Yellow Dead Cell Stain Kit (Invitrogen) was used to monitor cell death.
3
Skin Immune Profiling in Murine Model
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Mice (n = 9) were sacrificed on Day 3 and had their skin harvested for histological and flow cytometry analysis. Murine back skin sections were digested to assess immune cell populations as previously described34 (link). Briefly, precut skin sections were digested with Collagenase P (11213857001; Sigma-Aldrich), DNAse I (9003-98-9; Worthington) and Dispase II (D4693-1G; Sigma-Aldrich) in DMEM (Wisent Bio Products) for 1 h in 37°C. Cells were then filtered through 40 μm cell strainers before being stained for flow cytometry. Next, Fc receptors on cells were blocked with Mouse TruStain FcX (101319; BioLegend) and stained with cocktail combinations of: APC/Cy7 anti-CD45 (103116; Biolegend), PE/Cy5 anti-CD3 (100310; BioLegend), APC anti-CD8, PE/Cy7 anti-CD4, Alexa Fluora 700 anti-CD11b (101222; Biolegend), FITC anti-NKp46 (137605; Biolegend), PE anti-TIGIT, PerCP Cy5.5 anti-CD226, FITC anti-F4/80 (123108; Biolegend), APC anti-Ly6C (128015; Biolegend), and PE/Cy5 anti-CD11c (117316; Biolegend).
Additionally, skin samples were sectioned and stained with H&E for measuring epidermal thickness. Histology section images were captured, and epidermal thickness was measured using S-EYE 1.4 software. The epidermal thickness was quantified by measuring thickness in at least three distinct locations on each tissue section.
Additionally, skin samples were sectioned and stained with H&E for measuring epidermal thickness. Histology section images were captured, and epidermal thickness was measured using S-EYE 1.4 software. The epidermal thickness was quantified by measuring thickness in at least three distinct locations on each tissue section.
4
Memory CD4+ T Cell Isolation and Stimulation
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CD4+CD45RO+ T cells were isolated from PBMC using the immunomagnetic negative selection EasySep Human Memory CD4+ T Cell Enrichment Kit and Magnet (StemCell Technologies, Vancouver, Canada), following manufacturer provided indications. Recovery (40.9±14.1%) and purity (93.6±1.2%) of the enriched population was assessed by flow cytometry, staining with PE-Cy7 anti-CD4 (BioLegend, San Diego, CA, USA), FITC anti-CD3, PE-Cy5 anti-CD8, APC anti-CD45RA and PE anti-CD45RO (BD Biosciences, San Diego, CA, USA) antibodies.
For initial stimulation, memory CD4+ T cells were seeded in 96-well plates, in T cell medium, at 5×103 cells/well and incubated with 104 autologous irradiated PBMC and 2.5 μg/ml T. cruzi epimastigote lysate, or at 103 cells/well and incubated with 104 allogeneic irradiated PBMC from 3 non-related, non-infected donors, 1 μg/ml PHA and 50 IU/ml IL-2. This interleukin was also added on every culture since day 3, every 3 to 4 days, at a final concentration of 50 IU/ml. Half the medium (100 μl/well) was refreshed every 10 to 14 days, beginning at day 15.
For initial stimulation, memory CD4+ T cells were seeded in 96-well plates, in T cell medium, at 5×103 cells/well and incubated with 104 autologous irradiated PBMC and 2.5 μg/ml T. cruzi epimastigote lysate, or at 103 cells/well and incubated with 104 allogeneic irradiated PBMC from 3 non-related, non-infected donors, 1 μg/ml PHA and 50 IU/ml IL-2. This interleukin was also added on every culture since day 3, every 3 to 4 days, at a final concentration of 50 IU/ml. Half the medium (100 μl/well) was refreshed every 10 to 14 days, beginning at day 15.
5
Multiparametric Flow Cytometry Analysis of Tumor-Infiltrating Immune Cells
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Tumors were dissociated into single cells and were then filtrated through 70 μm cell strainers. Single cells were resuspended in cell staining buffer (#FXP005, 4abio) and then stained with fluorochrome-conjugated antibody combinations at appropriate concentration. The antibody information are shown as follows: PerCP-Cy5.5-Anti-CD45 (#103131, Biolegend), PE-Cy7-Anti-CD4 (#100421, Biolegend), FITC-Anti-CD8 (#ab237367, Abcam), APC-Anti-TNF-α (#506307, Biolegend), PE-Anti-IFN-γ (#505807, Biolegend), PerCP-Cy5.5-Anti-CD45R (#ab210342, Abcam), and PE-Anti-CD3 (#ab22268, Abcam). DAPI (#D9542, Sigma) was added to exclude dead cells. After washing, the stained cells were analyzed on a BD Fortessa machine. The data were processed with FlowJo software.
6
Multicolor Flow Cytometry for Immune Cell Profiling
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PE anti-CD3, Pacific Blue anti-CD8α, biotin-conjugated anti-CD8β, PE Cy7 anti-CD4, APC Cy7 anti-CD62L, APC anti-CD103, PerCP Cy5.5 anti-TCRγδ, PE Cy7 anti-TCRβ, APC Cy7-B220, PerCP Cy5.5 anti-B220, FITC-EpCAM, PE Cy7 anti-CD11b, APC anti-F4/80, biotin-conjugated anti-IgA antibodies were purchased from BioLegend. Streptavidin-PE was purchased from BD Biosciences.
7
Comprehensive Tumor Immune Cell Profiling
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Tumors were chopped into small pieces that were then transferred into gentleMACS Tubes (MACS Miltenyi Biotec), containing 10 mL of DMEM media and 1 mg/mL collagenase D (Sigma-Aldrich, COLLD-RO Roche, #11088866001). The tubes were placed on a gentleMACS Dissociator (MACS Miltenyi Biotec, #130-095-937) using the program 37_m_TDK2. After incubation, cells were filtered using 70 µm cell strainer and recovered by centrifugation. Cells were stained for live/dead with either LIVE/DEAD Fixable Violet Dead Cell Stain Kit, for 405 nm excitation (Thermo Fisher, cat#L34963) or Zombie NIR (BioLegend, cat#423105) then stained with a cocktail of surface mAbs Panel 1: BV711 anti-CD45 (BioLegend, cat#103147), PE anti-NK1.1 (BioLegend, cat#108707) and PE/Cy7 anti-CD8 (eBioscience, cat# 25-0083), APC anti-CD4 (eBioscience, cat#14-0042-81), BV421 anti-F4/80 (BioLegend, cat#123137) and PE/Dazzle 594 anti-CD183 (CXCR3) (BioLegend, cat#155914); Panel 2: BV711 anti-CD45 (BioLegend, cat#103147), APC anti-IFNg (BioLegend, cat#505810) and PE/Dazzle 594 anti-T-bet (BioLegend, cat#644828), PE/Cy7 anti-CD4 (BioLegend, cat#100422). After 30 min of staining, cells were washed and samples were run on FACS Symphony Cytometer (BD Biosciences). FlowJo V.10 was used for the analysis, cells were manually gated on size and granularity. Dead cells and doublets were excluded, and CD45 + cells were selected.
8
Splenocyte Proliferation Assay with CFSE
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The proliferation ability of splenocytes was evaluated by using the CFSE Cell Proliferation Assay Kit (Invitrogen). Splenocytes were stained with CFSE at 37 °C for 10 min, washed with complete Roswell Park Memorial Institute medium 1640 thoroughly, and then seeded into 24-well plates (1 × 106 cells/well) and incubated with H3 (5 μg/mL) for 60 h. The cells were then harvested and stained for 30 min at 4 °C with anti-mouse PE-Cy7-anti-CD4 and PE-Cy5-anti-CD8α antibodies (Biolegend) in the presence of Fc blocker. After washing and resuspending in flow cytometry staining buffer (PBS + 5% fetal calf serum), the cells were measured with a BD LSRFortessa flow cytometer (BD biosciences). Data were analyzed using the FlowJo software (FlowJo LLC). The splenocytes from naïve mice were used as control.
9
Isolation and Analysis of Intestinal Immune Cells
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Spleens were collected and mashed in 70 μm cell strainers with complete culture media. Red blood cells were lysed with RBC lysis buffer (eBioscience). To isolate LP cells, the small intestine was cut into pieces and digested by a buffer with Dispase, DNase, and Collagenase. Intestinal epithelial cells were removed, and Percoll was used for the isolation of lymphocytes. For surface staining, cells were blocked with anti-mouse CD16/32 (eBioscience), stained with fluorochrome-conjugated antibodies, and analyzed with a BD FACSAria II flow cytometer (BD Biosciences). For intracellular staining, a Foxp3 Fixation/Permeabilization kit (eBioscience) was used. Anti-mouse antibodies used in this study include APC anti-CD3, PE-Cy7 anti-CD4, FITC anti-CD8, PerCP-Cy5.5 anti-CXCR5, APC-Cy7 anti-PD-1, PE anti-B220, FITC anti-CD38, APC anti-GL7, FITC anti-IgA, PerCP-Cy5.5 anti-CD45, “Lin”: biotin anti-CD3 and biotin anti-CD19, a Zombie Aqua Fixable Viability Kit (Biolegend), RORγt-PE (BD Biosciences), and APC anti-biotin (Miltenyi Biotec). Flow cytometry data were analyzed with FlowJo.
10
Characterization of T-cell subsets in mouse eyeball
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The eyeball blood of mice was collected with EDTA anticoagulant tube on the 14th and 21st day after operation. Red blood cells were lysed by lysing buffer (BD Pharmingen, CA, USA) according to the manufacturer’s protocol. For cell surface staining, aliquots of single cell suspensions (1×106) were incubated with fluorophore-conjugated monoclonal antibodies at room temperature in the dark (Alexa Fluor 488-anti-CD3, PE-cy7-anti-CD4 or EF450-anti-CD4, PE-cy5.5-anti-CD8, BV421-anti-TCRγδ and/or APC-anti-CD25; all from Biolegend, San Jose, CA, USA). For intracellular staining, cells were stimulated by incubation for 4 h in RPMI 1640 medium, phorbol 12-myristate 13-acetate (50 ng/mL), ionomycin (1 µg/mL), and brefeldin A (1 µg/mL) (all from Biogems, Rocky Hill, NJ, USA) in a 5% CO2 atmosphere at 37 ℃. The cells were then washed with PBS, fixed, permeabilized, and stained with PE-cy7-anti-IL-17A (Biolegend, San Jose, CA, USA) according to the manufacturer’s protocol. For intranuclear transcription factor detect, cells were washed, fixed, permeabilized, and stained with PE-anti-Foxp3 (BD Biosciences, San Jose, CA, USA) according to the manufacturer’s protocol. Fluorescence data were collected on a FACS Aria Ⅲ (BD Biosciences, San Jose, CA, USA) and analyzed with FlowJo software (Tree Star, Ashland, OR, US).
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