Spleen cells from immunized mice (100 μl/well in 96 U-bottom plates) were incubated with individual peptides (adding 50 μl at 4 μg/ml for 2 h, and 50 μl Golgi Stop at 4 μl/ml for 4 h), in complete medium containing 10% FCS. Cells were then harvested, membrane stained using anti-mouse CD4-V500 (BD Biosciences), CD3-alexa488 (BD Biosciences), and CD8-PerCP (eBioscience) antibodies, and intracellular staining was performed using the intracellular staining kit (BD Biosciences) and anti-mouse IFN-γ-APC and TNF-α-eFluor 450 antibodies (eBioscience).
Cd8 percp
Manufactured by Thermo Fisher Scientific
Sourced in United States
The CD8-PerCP is a flow cytometry reagent that contains an antibody specific for the CD8 surface antigen, conjugated to the PerCP fluorescent dye. The CD8 antigen is a co-receptor expressed on the surface of cytotoxic T cells and a subset of natural killer cells. The PerCP dye allows for the detection and quantification of CD8-positive cells in flow cytometry applications.
Automatically generated - may contain errors
Lab products found in correlation
Cd3 apc, by Thermo Fisher Scientific (3 mentions)
Cd62l pe, by Thermo Fisher Scientific (2 mentions)
Cd45ra fitc, by Thermo Fisher Scientific (2 mentions)
Cd8 pe cy7, by Thermo Fisher Scientific (2 mentions)
Cd4 apc, by Thermo Fisher Scientific (2 mentions)
Ifn γ apc, by BD (2 mentions)
Cd4 percp, by Thermo Fisher Scientific (2 mentions)
Cd25 pe, by Thermo Fisher Scientific (2 mentions)
Accuri c6 flow cytometer, by BD (2 mentions)
Intracellular staining kit, by BD (1 mentions)
Cd3 alexa488, by BD (1 mentions)
Cd4 apc cy7, by BD (1 mentions)
Paraformaldehyde (pfa), by Merck Group (1 mentions)
C kit fitc, by Thermo Fisher Scientific (1 mentions)
Cd44 pe, by Thermo Fisher Scientific (1 mentions)
Cd62l fitc, by Thermo Fisher Scientific (1 mentions)
Rpmi 1640, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Atlanta Biologicals (1 mentions)
Collagenase type 1, by Worthington (1 mentions)
Cd69 pe cy7, by Thermo Fisher Scientific (1 mentions)
13 protocols using cd8 percp
1
Peptide-specific T-cell Response Assay
2
PBMC Membrane Antigen Staining
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PBMCs harvested from cultures were resuspended in 50 μl of FACS buffer and stained for 30 min on ice for respective membrane antigens using fluorochrome-conjugated monoclonal antibodies specific for human: CD4–APC-Cy7 (BD Pharmingen, San Diego, CA, USA) or CD8-PerCP (eBioscience, San Diego, CA, USA). After the washing step, cells were acquired and analyzed using a FACSCanto cell cytometer and the Diva software. Appropriate isotype controls were used in all experiments.
3
Stimulation and Intracellular Cytokine Analysis of T-cell Subsets
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Freshly isolated PBMCs (Day 0) and the expanded VSTs (Day 8) were resuspended at 1.0 × 106 cells per mL−1 and plated in 96 well plates (200 μL per well). The cells were then stimulated with 50 ng of PHA (positive control), RPMI alone (negative control) or viral peptides for 1 hour at 37 °C and for a subsequent 4 hours at 37 °C in presence of Brefeldin A (1 μg/mL). Following incubation, PBMCs and the expanded VSTs were washed twice with PBS and surface stained for 45 minutes with CD45RA FITC, CD62L PE and CD8 PerCP (ebioscience, San Diego, CA, USA) mAbs. The cells were then washed once more in PBS and fixed overnight at 4 °C in 4% Paraformaldehyde (Sigma Aldrich, St Louis, MO, USA). Excess fixating agent was washed off twice with permeabilization buffer (PBS+0.2% saponin) and cells were incubated for 45 minutes in permeabilization buffer and an anti-TNF-α mAb conjugated to APC (ebioscience, San Diego, CA, USA). Intracellular expression of TNF-α by the different T-cell subsets with and without peptide stimulation was analyzed on a BD accuri C6 flow cytometer.
4
Isolation and Analysis of Murine Immune Cells
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DMBA and BP were purchased from AccuStandard, Inc (New Haven, CT). RPMI 1640 was purchased from Sigma Chemical (St Louis, MO). Collagenase (Type 1) was purchased from the Worthington Biochemical Corp. (Lakewood, NJ). The CFU‐preB (M3630) media were obtained from StemCell Technologies (Vancouver, BC, Canada). Fetal bovine serum was obtained from Atlanta Biologicals (Flowery Branch, GA). The following monoclonal antibodies (mAbs) for FACS analysis of splenocyte and thymocyte cell populations were purchased from BD Pharmingen (San Jose, CA): CD45/B220‐Percp–, Gr‐1‐fluorescein isothiocyanate (FITC), CD4‐APC, CD8‐Percp, CD8‐PeCy7, CD62L‐FITC, and CD44‐PE, whereas F4/80 (FITC), Sca‐1(PE) and c‐kit (FITC) were obtained from eBioscience (San Diego, CA).
5
PBMC Immunophenotyping by Flow Cytometry
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For the CD4+ cells or PBMCs analysis, harvested cells were resuspended in 50 µl of fluorescent-activated cell sorting buffer and stained for 30 min on ice for respective membrane antigens using fluorochrome conjugated monoclonal antibodies specific for human: CD4–FITC, CD25-PE, human leukocyte antigen (HLA-) DR-PerCP (all from BD Pharmingen, San Diego, CA, USA), CD8-PerCP, and CD69-Pe-Cy7 (both from eBioscience, San Diego, CA, USA). To assess viability, PBMCs were stained with 7-aminoactinomycin D (7AAD). After the washing step, cells were acquired and analyzed using a FACSCanto cell cytometer and Diva software. Appropriate isotype controls were used in all experiments. The gating strategy and applied isotype controls are shown in Supplemental Fig. S1.
6
Multiparametric Immune Cell Analysis
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Spleens, mesenteric lymph node (LN), and livers from WT and iNOS-KO rats were removed aseptically and dispersed through a 70-μm nylon strainer. Single-cell suspensions of peripheral blood, spleens, LN, and livers were prepared by Ficoll-Hypaque (Hao Yang Biological Manufacture, Tianjin, China) gradient centrifugation, washed twice, and suspended in PBS containing 0.1% BSA and 0.05% sodium azide. For surface staining, 2 × 106 cells per 100 μl were incubated with CD3-APC (eBioscience, CA), CD19-FITC (Abcam, UK), CD4-PerCP-eFluor710 (eBioscience, CA), CD8-PE-Cy7 (eBioscience, CA), and macrophage marker-PE (eBioscience, CA) for 30 min at 4°C in the dark. For the detection of intracellular cytokines, cells in complete RPMI 1640 medium were stimulated with PMA (20 ng/ml), Ionomycin (1 μg/ml), and Brefeldin A (10 μg/ml) (Sigma-Aldrich, Germany) for 6 h. Surface molecule staining CD3-FITC (BD Biosciences, USA), CD4-PE-Cy7 (eBioscience, CA), and CD8-PerCP (eBioscience, CA) for 30 min at 4°C was carried out. Cells were fixed in 0.4% paraformaldehyde for 20 min, permeabilized with 0.1% saponin buffer (Sigma-Aldrich, Germany), and stained for IFN-γ-APC (BD Biosciences, USA) and IL-4-PE (BD Biosciences, USA). The expression of surface molecules and intracellular molecules were analyzed on a BD LSR II flow cytometer using FlowJo v.8 software (Treestar, CA).
7
Multiparameter Flow Cytometry Analysis
8
Phenotyping T-cell Subsets by Flow Cytometry
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Freshly isolated PBMCs and the expanded VSTs (1.0 × 106) were labeled with pre-diluted monoclonal antibodies (mAbs) in a 4-color direct immunofluorescence assay. Cells were incubated at room temperature in the dark for 45-minutes. The mAb combinations consisted of CD45RA FITC, CD62L PE, CD4 PerCP or CD8 PerCP and CD3 APC (ebioscience, San Diego, CA, USA). This allowed for the determination of naïve (NA; CD45RA+/CD62L+), central memory (CM; CD45RA−/CD62L+), effector memory (EM; CD45RA−/CD62L−) and CD45RA+ EM (EMRA; CD45RA+/CD62L−) subsets within the CD4+ and CD8+ T-cells29 (link)30 (link) before and after expansion. The phenotypes of the freshly isolated PBMCs (Day 0) and the expanded VSTs (Day 8) were assessed on a BD Accuri C6 flow cytometer (Accuri, Ann Arbor, MI, USA) as previously described22 (link). The total number of T-cell subsets in blood before and after exercise was determined by multiplying the percentage of cells staining positive for the appropriate surface markers in the flow cytometry lymphocyte gate by the total blood lymphocyte count (Table 2 ).
9
Multicolor Flow Cytometry of Immune Checkpoint Targets
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Fluorescently-conjugated antibodies CD3-e450, CD8-PerCP, CD4-FITC, CD4-e450, CD4-PerCP, CD25-APC, were purchased from Ebioscience (San Diego, CA). CD8-PE-TxRD was purchased from Invitrogen (Carlsbad, CA). Therapeutic anti-CTLA4 (clone 9D9 or UC10), anti-OX40 (clone OX86), anti-CD40 (clone FGK4.5), anti-CD4 (clone GK1.5), and anti-CD25 (clone PC.61.5.3) antibodies were obtained from BioXcell (Branford, CT) and resuspended in sterile PBS to a concentration of 1mg/mL. Antibodies were administered as 250μg (anti-OX40 and anti-CTLA4) or 100μg (anti-CD4 and anti-CD25) intraperitoneally. DEC205ova was kindly provided by CellDex Therapeutics (Hampton, NJ). SIINFEKL-Kb tetramers were obtained from the NIH Tetramer Core Facility at Emory University (Atlanta, GA).
10
Multiparametric Flow Cytometry Assay
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