For surface marker studies (CD4, CD8α and/or CD8β), cells were assayed at the indicated time point post gRNA transduction. Cells were stained with either CD4-PE (clone GK1.5, in-house, 1:800) or CD8a-PE (clone 53-6.7, BioLegend, #100707, 1:800) or CD8b-APC/Cy7 (clone YTS156.7.7, BioLegend, #126619, 1:600) and analysed with BD FACSymphony A3 or BD LSRFortessa and subsequently using FlowJo 10.4.1. For Dreg1, Cd3e, Cd3d, Cd3g, Cd8b, Hba-x and Hbb-y studies, cells were sorted on BD FACSAria Fusion or FACSAria III 7 days post gRNA transduction. SAM cells were sorted as mCherry+ BFP + TagRFP657+ population. SunTag-VP64 or SunTag-TET1 cells were sorted as BFP + GFP + TagRFP657 + population. TETact v1-v3 cells were sorted as BFP + GFP + mCherry + TagRFP657 + population. Gating strategies are shown in Supplementary Fig. 9 .
Cd8b apc cy7
Manufactured by BioLegend
CD8b-APC/Cy7 is a fluorochrome-conjugated antibody that binds to the CD8b subunit of the CD8 co-receptor on the surface of T cells. It can be used to identify and study CD8-positive T cell populations in flow cytometry applications.
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Lab products found in correlation
Cd4 pe, by BioLegend (1 mentions)
Cd8a pe, by BioLegend (1 mentions)
Facsaria fusion, by BD (1 mentions)
Facsymphony a3, by BD (1 mentions)
Monesin, by Thermo Fisher Scientific (1 mentions)
Anti cd28, by Thermo Fisher Scientific (1 mentions)
Cd45 percp cy5, by BioLegend (1 mentions)
Cd44 bv421, by BioLegend (1 mentions)
2 protocols using cd8b apc cy7
1
Flow Cytometry Analysis of Immune Cells
2
Alloantigen-Primed T Cell Phenotyping
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Alloantigen-primed IFN-γ and TNFα-producing T cells were analyzed using FACS. Briefly, splenocytes were prepared and incubated in stimulating media (25 000 000 cells/well). The stimulating media contains anti-CD28 (2 μg/mL), Brelfeldin (ThermoFisher) and Monesin (ThermoFisher). The splenocytes were divided into three groups and treated with stimulating media containing firefly luciferase (negative control group, 1 mg/mL), LacZ (test group, 1 mg/mL), p15E (KSPWFTTL, test group, 1 mg/mL) or PMA/ionomycin mix (positive control group). The incubation started with for 5 hours in 37°C and then transfer to 4°C overnight. After the incubation, the splenocytes were first stained for surface marker (Biolegend) and the following panel was used: CD4 PE-TR (#1005666), CD8b APC-CY7 (#126620), CD45 PerCP-CY5.5 (#123128) and CD44 BV421 (#103039). The cells were fixed and permeabilized after surface staining, then followed by staining for IFNγ PE-Cy7 (#505826) and TNFα AF647 (#506314).
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