Anti cd95 fitc

Evaluation of T cell surface markers was performed via immunofluorescence staining and analyzed via flow cytometry. Monoclonal antibodies (mAbs) used to characterize transduced T cells include: anti-CD3-APC Cy7, anti-CD4-APC, anti-CD8-PerCP/Cy5.5, anti-CD34-PE, anti-CCR7-BV785, anti-CD45RA-PE Cy7 (Biolegend, San Diego, CA), and anti-CD62L-BV650, anti-CD95-FITC (BD Biosciences). Flow cytometry was performed using the LSR Fortessa flow cytometer (BD Biosciences) and data was analyzed with FlowJo software V10.1.

Expression of γH2AX was measured using flow cytometry. Cells were irradiated (1Gy or 5Gy) and at indicated times, cells were permeabilized (Foxp3 staining kit; eBioscience) and stained using the following antibodies: isotype-AF488 (BD Biosciences #557782) or γH2AX-AF488 (phosphorylated-H2AX-ser-139; Cell Signaling #9719S). CD95 expression on NALM-6 cells was determined by flow cytometry using anti-CD95-FITC (BD biosciences #555673) following irradiation (1Gy or 5Gy) and 16 h culturing. Data were normalized for isotype control (isotype-AF488).

To assess the expression of CD25, CD95, and CD69 on CD4⁺ and CD8⁺ T cells, the cells (1.5 × 10⁶/mL) were stimulated for 24 or 48 h at 37 °C with ArtinM (1.25 μg/mL) or PMA (50 ng/mL) plus ionomycin (1 μM), then washed and incubated with Fc block (10 μg/mL) for 30 min. Afterwards, the cells were washed again and incubated for 45 min with anti-CD25 PE (10 μg/mL, clone 3C7; BD Biosciences), anti-CD95 FITC (10 μg/mL, clone Jo2; BD Biosciences), or anti-CD69 FITC (10 μg/mL, clone H1.2F3; BD Biosciences). The cells were then washed with PBS and resuspended in PBS containing 0.5% formaldehyde. The frequency of fluorescent cells was analyzed by flow cytometry (Guava^® easyCyte).
The frequency of dead cells was assessed by analyzing the Annexin V binding and propidium iodide (PI) incorporation by CD4⁺, CD8⁺, and Jurkat T cells. They were incubated with ArtinM (concentrations specified in the figure legends), staurosporine (1 μM), As₂O₃ (3 μM), or medium alone and then stained with Annexin V-FITC (for 40 min) and PI (for an additional 5 min). The fluorescence emission due to the reaction of cells with Annexin V and PI was quantified using a Guava^® easyCyte flow cytometer.