T cells were isolated from tumor single cell suspensions by antibody staining followed by cell sorting on a 5-laser FACSAria Fusion sorter (Stanford FACS Facility) purchased using funds from the Parker Institute for Cancer Immunotherapy. Tumor cell suspensions were stained in PBS with Zombie Aqua dye (Biolegend) for viability assessment. This was followed by staining in PBS with 2% FBS in Fc Blocking solution (Biolegend) plus the following antibodies: anti-CD4 (OKT4, Biolegend), anti-CD8 (SK1, Biolegend), anti-CD3 (OKT3, Biolegend), anti-CD45 (H130, Biolegend), anti-CD25 (BC96, Biolegend), anti-PD-1(EH12.2H7, Biolegend), anti-CD137 (4B4-1, BD Biosciences), anti-HLA-DR (L243, Biolegend). CD3+CD45+AquaZombie- cells were index sorted directly into 96-well plates preloaded with 4 uL of capture buffer, snap frozen on dry ice, and stored at −80°C. Ectopic HLA-B∗35 was detected with anti-HLA-BC monoclonal antibody (clone B1.23.2, Thermo Fisher Scientific). Transduced Jurkat 76 cells expressing exogenous TCRα/β chains were sorted on a FACSAria Fusion sorter at Stanford or a BD Biosciences Influx High Speed Cell Sorter at the Flow Cytometry Core Facility of the Cancer Institute of New Jersey.
Anti cd8 sk1
Manufactured by BioLegend
Anti-CD8 (SK1) is a mouse monoclonal antibody that binds to the CD8 antigen. CD8 is a glycoprotein expressed on the surface of cytotoxic T cells, which play a key role in the adaptive immune response.
Automatically generated - may contain errors
Lab products found in correlation
Anti cd3 okt3, by BioLegend (2 mentions)
Anti cd4 okt4, by BioLegend (2 mentions)
Fc blocking solution, by BioLegend (1 mentions)
Anti cd137 4b4 1, by BD (1 mentions)
Anti cd45 h130, by BioLegend (1 mentions)
Anti cd25 bc96, by BioLegend (1 mentions)
Anti pd 1 eh12.2h7, by BioLegend (1 mentions)
Zombie aqua dye, by BioLegend (1 mentions)
Influx high speed cell sorter, by BD (1 mentions)
Anti hla dr l243, by BioLegend (1 mentions)
Facsaria fusion sorter, by BD (1 mentions)
Hla dr l243, by BioLegend (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Ma900 sorter, by Sony (1 mentions)
Dynabeads human t activator cd3 cd28, by Thermo Fisher Scientific (1 mentions)
Intracellular ic fixation buffer, by Thermo Fisher Scientific (1 mentions)
Qa16a02, by BioLegend (1 mentions)
Golgistop, by BD (1 mentions)
Permeabilization buffer, by Thermo Fisher Scientific (1 mentions)
Anti cd3 ucht1, by BioLegend (1 mentions)
4 protocols using anti cd8 sk1
1
Single-Cell Sorting of Tumor-Infiltrating T Cells
2
Flow Cytometry and Functional Assays
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The following antibodies were used for flow cytometric analysis: anti-TCR (clone IP26; BioLegend), anti-CD3 (OKT3; BioLegend), anti-vβ3.1 [JOVI.3; Becton, Dickinson & Company (BD)], anti-CD4 (OKT4; BioLegend), anti-CD8 (SK1; BioLegend), HLA-A2 (BB7.2; BioLegend), and HLA-DR (L243; BioLegend). Isotype-matched control antibodies were used in all cases.
For functional reporter assays, blinatumomab was obtained from Myonex; staphylococcal enterotoxin E was obtained from Toxin Technology Inc.; recombinant H3 was obtained from Sino Biological; 2020 Fluzone Quadrivalent (NDC 49281-420-88) was obtained from Sanofi Pasteu.
Antigenic peptides [MART-126–35, MART-127–35, MART-1Leu26–35, hemagglutinin (HA)307–319, and associated HA peptides from H3, H1, and H5] were custom synthesized at >95% purity by Vivitide, and lyophilized peptides were then reconstituted in dimethyl sulfoxide (Sigma-Aldrich) or ddH20, and stored at −80°C.
For functional reporter assays, blinatumomab was obtained from Myonex; staphylococcal enterotoxin E was obtained from Toxin Technology Inc.; recombinant H3 was obtained from Sino Biological; 2020 Fluzone Quadrivalent (NDC 49281-420-88) was obtained from Sanofi Pasteu.
Antigenic peptides [MART-126–35, MART-127–35, MART-1Leu26–35, hemagglutinin (HA)307–319, and associated HA peptides from H3, H1, and H5] were custom synthesized at >95% purity by Vivitide, and lyophilized peptides were then reconstituted in dimethyl sulfoxide (Sigma-Aldrich) or ddH20, and stored at −80°C.
3
WNK3 Knockdown in T Cell Subsets
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For WNK3 knockdown in human CD4+ and CD8+ T cells, live CD4+ and CD8+ T cells were sorted from human PBMCs using an MA900 sorter (SONY, San Jose, CA, USA) and stimulated with plate-bound anti-human CD3 (#BE0001-2, BioXCell, Lebanon, NH, USA)/anti-human CD28 (#BE0248, BioXCell) and 50 IU IL-2 for 24 h. These cells were spin-infected (900 × g, 90 min, 37 °C) with lentiviral supernatant (shNC and shWNK3). Four days after lentiviral infection, cells were selected with 1 μg/ml puromycin for 7 days. Then, the cells were stimulated with Dynabeads Human T-Activator CD3/CD28 (Gibco) for 36 h. BD GolgiStop (#554724, BD Biosciences) was added for the last 6 h of stimulation. After stimulation, the cells were washed, and surface molecules were stained with anti-CD4 (OKT4, BioLegend) and anti-CD8 (SK1, BioLegend). Cells were then fixed with eBioscience/Invitrogen Intracellular (IC) Fixation Buffer (#00-8222-49, Invitrogen), washed with 1x Permeabilization Buffer (#00-8333-56, Invitrogen) and stained with antibodies for anti-perforin (B-D48, BioLegend) and anti-granzyme B (QA16A02, BioLegend).
4
Multiparametric Flow Cytometry Immune Profiling
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PBMCs were isolated using Ficoll-Paque density gradient centrifugation, frozen in freezing medium [10% DMSO, 90% fetal bovine serum (FBS)] and stored at -80°C until use. For staining, PBMCs were thawed using complete RPMI (RPMI+10%FBS) and washed with PBS. The cells were first stained using Fixable Aqua Dead Cell Kit (Thermofisher), followed by staining with anti-CD3 (UCHT1) (Biolegend), anti-CD4 (RPA-T4) (BD Biosciences), anti-CD8 (SK1) (Biolegend), anti-CD19 (HIB19) (BD Biosciences), anti-CD27 (O323) (Biolegend), anti-CD56 (B159) (BD Biosciences), anti-CD45RA (2H4) (Beckman Coulter), anti-CD38 (HIT2) (BD Biosciences) and anti-CD38 (JK36) (Beckman Coulter). The cells were washed and analyzed using BD LSRFortessa™ cell analyzer. Data analyses were performed using Flowjo V10.5.3 (BD)
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