For assessing early TCR-driven signaling, J76-CD8 stably expressing TCRs were stimulated with peptide-pulsed HLA-A2-tranfected T2 cells65 . T2-A2 cells were loaded with a single dose (1 µM) M1-peptide or with varying concentration of M1-peptide (10−12 M to 10−7 M) for 1 hr at 37 °C. Unloaded excess M1-peptide was removed by washing cells twice with PBS. M1-loaded T2-A2 cells (0.1×106 cells) were incubated with TCR expressing J76-CD8 cells (0.4×106 cells) at 37°C in 24-well dishes for 12 hr. After 12 hr incubation, cells were cooled on ice and harvested for FACS staining. 0.2–0.5 million cells were stained with anti-CD69 antibody (BioLegend, FN50), anti-TCR antibody BioLegend, IP26), and LIVE/DEAD® Fixable Violet dye for 30 min 4°C. Stained cells were washed with PBS and analyzed by FACS as described above.
Live dead fixable violet dye
Manufactured by BioLegend
The LIVE/DEAD® Fixable Violet dye is a fluorescent stain used to discriminate between live and dead cells. It is a cell-impermeant dye that can only enter cells with compromised membranes, which are typically dead or dying cells. The dye binds to cellular amines, producing a bright fluorescent signal in dead cells. This allows for the identification and quantification of live and dead cells in a sample.
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TCR-driven Signaling Assay Protocol
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TCR-driven Signaling Assay Protocol
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For assessing early TCR-driven signaling, J76-CD8 stably expressing TCRs were stimulated with peptide-pulsed HLA-A2-tranfected T2 cells65 . T2-A2 cells were loaded with a single dose (1 µM) M1-peptide or with varying concentration of M1-peptide (10−12 M to 10−7 M) for 1 hr at 37 °C. Unloaded excess M1-peptide was removed by washing cells twice with PBS. M1-loaded T2-A2 cells (0.1×106 cells) were incubated with TCR expressing J76-CD8 cells (0.4×106 cells) at 37°C in 24-well dishes for 12 hr. After 12 hr incubation, cells were cooled on ice and harvested for FACS staining. 0.2–0.5 million cells were stained with anti-CD69 antibody (BioLegend, FN50), anti-TCR antibody BioLegend, IP26), and LIVE/DEAD® Fixable Violet dye for 30 min 4°C. Stained cells were washed with PBS and analyzed by FACS as described above.
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