Characterization of PBMCs by Flow Cytometry

PBMCs were stained with the following surface and intracellular antibodies: LIVE/DEAD® Fixable Aqua Dead Cell Stain Kit, (ThermoFisher Scientific), to exclude dead cells, CD3 (SK7), CD4 (RPA-T4), CD8 (SK1 and RPA-T8), CTLA-4 (BNI3), TIM-3 (F38-2E2), CD244 (C1.7), CD71 (M-A712), LAG-3 (3DS223H), CD160 (BY55), PD-1 (EH12.1), PDL-1 (M1H1), PDL-2 (MIH18), CD11b (ICRF44), CD14 (M5E2) and Gal-9 (9M1-3) (BD Biosciences), TIGIT (MBSA43, eBioscience). Following staining cells were fixed with 4% paraformaldehyde, before analysis. Cells were acquired using an LSR FortessaSORP flow cytometer (BD Biosciences) and analysed with FlowJo software (Ashland, Oregon, USA). For CFSE labelling, PBMCs were labelled with 1.25μM CFSE (ThermoFisher Scientific) as previously described [82 (link)] before stimulation with SEB or a-CD3/CD28.

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Okoye I., Namdar A., Xu L., Crux N, & Elahi S. (2017). Atorvastatin downregulates co-inhibitory receptor expression by targeting Ras-activated mTOR signalling. Oncotarget, 8(58), 98215-98232.

Publication 2017

LIVE/DEAD® Fixable Aqua Dead Cell Stain Kit TIGIT (MBSA43, LSR FortessaSORP flow cytometer CFSE

Antibodies Cd11b Cd71 Cells Cfse Ctla 4 Gal 9 Intracellular Paraformaldehyde Stain Tigit Tim 3

Corresponding Organization :

Other organizations : University of Alberta

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Variable analysis

independent variables

Stimulation with SEB or a-CD3/CD28

dependent variables

Expression of surface and intracellular markers: CD3, CD4, CD8, CTLA-4, TIM-3, CD244, CD71, LAG-3, CD160, PD-1, PDL-1, PDL-2, CD11b, CD14, Gal-9, TIGIT
Cell proliferation as measured by CFSE labeling

control variables

Live/dead cell exclusion using LIVE/DEAD® Fixable Aqua Dead Cell Stain Kit
Staining and fixation protocols
Flow cytometer and analysis software

controls

Positive control: Not specified
Negative control: Not specified

Annotations

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