Splenocytes from intramuscularly vaccinated mice were incubated in culture with a pool of 53 overlapping 15-mer SARS-CoV-2 RBD peptides (jpi: PM-WCPV-S-RBD) for 6 h at 37 °C. Following blocking with 0.1% BSA-PBS, cells were stained on ice with CD4 APC/Cy7 (Biolegend: 100413), CD8 PerCP (Biolegend: 100713), and Zombie NIR Fixable Viability Kit (Biolegend: 423105). Stained cells were fixed and permeabilized with the Cyto-Fast™ Fix/Perm Buffer Set (Biolegend: 426803). Subsequently, intracellular staining was performed with anti-IFN-γ Brilliant Violet 421 (Biolegend: 505829) and anti-TNF-α PE (Biolegend: 506305). Analysis was performed on a BD FACSCalibur, using FlowJo X10.0 software.
Cd8 percp
Manufactured by BioLegend
Sourced in United States, Germany
The CD8-PerCP is a monoclonal antibody conjugated with the PerCP (Peridinin Chlorophyll Protein) fluorescent dye. It is designed for the identification and enumeration of CD8+ T cells in flow cytometry applications.
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Lab products found in correlation
Cd3 fitc, by BioLegend (4 mentions)
Cd4 apc, by BioLegend (4 mentions)
Cd4 fitc, by BioLegend (3 mentions)
Cd3 apc cy7, by BioLegend (3 mentions)
Ifn γ pe cy7, by BD (3 mentions)
Lsrfortessa, by BD (3 mentions)
Foxp3 apc, by Thermo Fisher Scientific (2 mentions)
Cd44 apc, by BioLegend (2 mentions)
Ifn γ apc, by BioLegend (2 mentions)
Il 17 pecy7, by BioLegend (2 mentions)
Cytofix cytoperm, by BD (2 mentions)
Facscalibur, by BD (2 mentions)
Golgistop, by BD (2 mentions)
Live dead fixable blue dead cell stain kit, by Thermo Fisher Scientific (2 mentions)
Tnf α fitc, by BD (2 mentions)
Il 2 bv711, by BD (2 mentions)
Il 21 pe, by Thermo Fisher Scientific (2 mentions)
Cd4 apc cy7, by BD (2 mentions)
Perforin pe dazzle 594, by BioLegend (2 mentions)
Cd69 bv650, by BD (2 mentions)
30 protocols using cd8 percp
1
SARS-CoV-2 RBD-specific T-cell Response Assay
2
CD8+ T cell depletion and HIV-1 p24 expression
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Peripheral blood mononuclear cells were depleted of CD8+ T cells (StemCell Technologies, Vancouver BC, Canada) and cultured in lymphocyte medium without phenol red supplemented to 50 IU mL−1 IL‐2 (NCI) for up to 144 h. Aliquots were removed at 24‐h intervals and stained with anti‐human CD4 (PE‐Vio770), CD3 (VioGreen), from Miltenyi, CD8 (PerCP, Biolegend), PVR (APC, Invitrogen) and intracellular anti‐HIV‐1 p24 (FITC, Santa Cruz) using the Inside Stain Kit (Miltenyi).
3
Flow Cytometric Analysis of Lymphocyte Subsets
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Cell suspensions from iliac lymph nodes, blood and tumors were first incubated with 1µg/106 cells of Fc block treatment (BD Pharmigen) and then stained for surface markers in FACS buffer (PBS + 1% FBS). For Treg staining on cells isolated from iliac lymph nodes: Live/Dead (Fixable Near-IR Dead Cell stain kit, Life Technologies); CD3 FITC, clone 145-2C11 (Biolegend); CD4 Pacific Blue, clone RM4-5 (Biolegend); Foxp3 APC, clone FJK-16S (eBioscience). For Treg staining on cells isolated from tumors: Live/Dead (Fixable Near-IR Dead Cell stain kit, Life Technologies); CD45 PerCP-Cyanine 5.5 (eBioscience); CD4 Pacific Blue, (Biolegend); CD8 Brilliant Violet 510, clone 53-6.7 (Biolegend); Foxp3 APC, (eBioscience). For effector memory staining on cells isolated from blood: Live/Dead (Fixable Near-IR); CD8 PerCP, (Biolegend); CD44 APC (Biolegend); CD62L FITC, clone MEL-14 (BD Pharmigen).
4
Checkpoint Blockade Immunotherapy Protocols
5
Bladder Immune Cell Profiling
6
Flow Cytometry Analysis of Lung Immune Cells
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BAL and mLN cells were processed and stained for flow cytometry as previously described [19] (link), [21] (link) with the following antibodies: CD16/32 (Fc blocker), CD4 (FITC), CD25 (PE-Cy7), and Foxp3 (APC) from eBioscience (San Diego, CA); CD3 (APC/Cy7) and CD8 (PerCP) from BioLegend (San Diego, CA). The MHC class I tetramer (PE) specific for H-2Db-resricted epitope of the influenza nucleoprotein (NP, DbNP366-74) of PR/8 was obtained from the NIH Tetramer Core Facility (Atlanta, GA). Stained cells were subsequently analyzed on a CyAn ADP Analyzer flow cytometer (Beckman Coulter, Inc., Fullerton, CA), and all flow cytometry data were analyzed via FlowJo Software (TreeStar, Ashland, OR). All flow cytometry analysis of T cells consisted of a doublet exclusion gate followed by gating of CD3+ cells for further analysis of CD4+ and CD8+ T cell populations.
7
Flow Cytometric Analysis of HLA Tetramers and Intracellular Cytokines
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All flow cytometric measurements were performed on a FACSCanto II (BD) and evaluated using FlowJo version 9.2 (Tree Star, Ashland, OR, USA). For HLA tetramer analysis, T cells were washed in FACS buffer containing 2% FCS, 2 mM EDTA (Carl Roth, 8040) and 0.02% sodium azide (Merck Millipore, 8223350100) in PBS, followed by a staining for dead cells using Live/Dead fixable Aqua dead cell stain kit (Invitrogen, L34957). Afterwards, cells were washed and incubated with 2.5 µg/mL of the respective tetramer in tetramer solution (FACS buffer with 50% FCS) for 30 min at RT. Finally, cell surface molecules were stained with CD8-PerCP (Biolegend, 301030) for 20 min at 4°C.
Intracellular cytokine staining was performed by incubating in vitro primed T cells with 10 µg/mL candidate or control peptide, Golgi-Stop-Solution (BD Bioscience, 554724) and anti-CD107a-FITC antibody (BD Bioscience, 555800) for 6 h. Afterwards, cells were stained for dead cells using Live/Dead fixable Aqua dead cell stain kit followed by staining with CD8-PerCP for 20 min. Intracellular staining was performed after 30 min permeabilization with Cytoperm/Cytofix solution (BD Bioscience, 554722) using the following antibodies: IFNγ-PE-Cy7, IL-2-APC, TNFα-PacificBlue and MIP-1β-PE (all BD Bioscience, 557844, 554567 and 550078; except TNFα, Biolegend, 502920).
Intracellular cytokine staining was performed by incubating in vitro primed T cells with 10 µg/mL candidate or control peptide, Golgi-Stop-Solution (BD Bioscience, 554724) and anti-CD107a-FITC antibody (BD Bioscience, 555800) for 6 h. Afterwards, cells were stained for dead cells using Live/Dead fixable Aqua dead cell stain kit followed by staining with CD8-PerCP for 20 min. Intracellular staining was performed after 30 min permeabilization with Cytoperm/Cytofix solution (BD Bioscience, 554722) using the following antibodies: IFNγ-PE-Cy7, IL-2-APC, TNFα-PacificBlue and MIP-1β-PE (all BD Bioscience, 557844, 554567 and 550078; except TNFα, Biolegend, 502920).
8
Multiparametric Flow Cytometry Analysis
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Peripheral blood (PB) samples were obtained, stained for multiparametric flow cytometry, and fractionated in 4 Eppendorf tubes (A, B, C, and D) containing 500 μL of blood each. The sample in tube A was unstimulated; tube B was stimulated with brefeldin A plus monensin; tube C was stimulated with 40 ng PMA plus 1 μg ionomycin; tube D was stimulated with 40 ng of PMA and 1 μg ionomycin plus brefeldin A and monensin. Samples were incubated at 37°C in a 5% CO2 atmosphere for 6 h. The following antibodies were used for cell surface staining: mouse anti-human CD4 PE-TexRed or CD4 PerCP, CD8 PE-TexRed, CD8-PErCP, CD25-APC (BioLegend, San Diego, CA), CD8-FITC, CD8-PE, or CD3-PerCP (BD, San Jose, CA). Cells were incubated for 20 min at 4°C in staining buffer (PBS, 0.5% BSA, and 0.01% sodium azide). The cell suspensions were then washed, lysed, and permeabilized for intracellular staining with anti-human-TGFβ-FITC, Bcl-2-FITC, active caspase 3-FITC, Ki67-PE-Cy7, Ki67-PerCP, IFNγ-APC, IL17-FITC, and IL17-PE-Cy7 (BioLegend). The tubes were incubated for 20 min at 4°C in the dark. Finally, the cells were washed, fixed with 1% paraformaldehyde, and analyzed in a flow cytometer (FACSAria III, BD) (Figure 1 ).
9
Flow Cytometric Analysis of CTLA-4 Expression
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Fasting, EDTA-anticoagulated blood samples were collected. The flow cytometric analysis was performed as described in detail in our prior publication [20 (link)] using the following flow cytometric antibodies: CD3-PE-Cy7, CD8-PerCP, CD25-AF700 (Biolegend, San Diego, CA, USA), Foxp3-PE-CF594 and CTLA-4-APC (BD Biosciences, San Jose, CA, USA). A Beckman Coulter Navios flow cytometer and version 1.2 of Beckman Coulter Kaluza software (Brea, CA, USA) were used for quantitative analysis. CTLA-4 expression levels were assessed intracellularly; median fluorescence intensity values (MFI) are indicated. Our gating strategy is described in Figure 1 .
10
Cytokine Profiling of Activated PBMCs
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