Propidium iodide ready flow reagent

Leaky or damaged cells were assessed by flow cytometry using Propidium Iodide Ready Flow Reagent as per manufacturer’s instructions (Invitrogen) with minor alterations. Samples were prepared as for membrane potential assay in sterile PBS and two drops of the PI Ready Flow reagent were added per sample and left to incubate at room temperature for 15 min. Red Fluorescence (FL3 channel) was recorded using the BD Biosciences Accuri C6 flow cytometer (BD Biosciences, USA). At least three biological replicates were assayed per condition.

The following antibodies from BioLegend were diluted in flow cytometry buffer (DPBS, 2% FBS, 2 mM EDTA): PerCP-Cy5.5-conjugated anti-human CD2 (RPA-2.10, 0.25 μg ml⁻¹, #300216), PE-Cy7-conjugated anti-human CD3e (UCHT1, 0.75 μg ml⁻¹, #300420), Pacific Blue or PE-Cy7-conjugated anti-human CD20 (2H7, 1 μg ml⁻¹, #302320 or #302312), APC-Cy7 or PE-Cy7-conjugated anti-human CD69 (FN50, 0.5 μg ml⁻¹, #310914 or #310912), and APC anti-HA.11 epitope tag (16B12, 0.5 μg ml⁻¹, #901524). BV395-conjugated CD20 (2H7, 0.25 μg ml⁻¹, #563782) was purchased from BDbiosciences. Propidium Iodide Ready Flow Reagent (Thermo Fisher, #R37169) was used as viability dye. GFP and BFP were additionally analyzed to identify TCR activation and minigene translation efficiency. Cells were washed before staining for 20 min at 4°C in the dark. Samples were then washed and analyzed using a BD FACSAria Fusion or BD FACSAria II cell sorter, or in high throughput using a Sony ID7000 spectral analyzer. Data analysis was performed using FlowJo (10.6.1). For single-variable analysis, the percentage of positive cells was calculated as the integrated area under the histogram not overlapping with control samples using FlowJo’s population comparison (Overton) method.

After cell synchronization using the double thymidine block, cells were trypsinized and quenched with media then centrifuged at 2,000 rpm for 5 min. The cells were then resuspended in 75% ethanol for at least 1 h at −20°C for fixation. The cells were centrifuged at 4,000 rpm for 2 min and then resuspended in PBS containing 0.25% Triton X-100 for 15 min. Cells were centrifuged at 4,000 rpm for 2 min and resuspended in PBS containing 10 μg/ml RNase A (Qiagen) and Propidium Iodide Ready Flow Reagent (ThermoFisher Scientific). Subsequent detection of the cell cycle phase distribution was accomplished by using propidium iodide for nuclear staining and detection using the BD FACSymphony A3 flow cytometer and collecting FSC, SSC, PE for propidium iodide, and BB515 for compensation with gating for single cells. The resulting data was analyzed by FlowJo software.