Modfit lt version 3.3

Two days after activation and drug treatment splenocytes were harvested, washed with PBS followed by resuspension in PBS with 2% FBS. Cell cycle analysis was performed taking advantage of PI staining using an established protocol^{49 (link)}. Cells were washed in cold PBS and resuspended in PI/Triton X-100 staining solution (10 ml 0.1% (v/v) Triton X-100 in PBS with 2 mg DNAse-free RNAse A and 0.4 ml of 500 µg/ml PI), followed by incubation at 4 °C for 30 min. Stained cells were analyzed on a FACSCalibur™ with the software CellQuest™ (BD Biosciences). Cell cycle analysis was conducted using the software ModFit LT, version 3.3 (Verity Software House Inc.). Cells were identified by gating into the lymphocyte population, followed by single cell gating to exclude doublets and aggregates. This was followed by identification of the G0/G1 population and processing with the software ModFit LT, version 3.3 (Verity Software House Inc.) to calculate the percentage of cells in different cell cycles.

Cell cycle analysis was performed by measuring cellular DNA content using FACS. Cells were dissociated using accutase and washed twice with PBS. 1 × 10⁶ cells/ml cells were resuspended in PBS and supplemented with 70% (v/v) absolute ethanol while gently vortexing at half speed. Cells were then incubated on ice for 15 minutes and subsequently stored at −20 °C until staining for flow cytometry analysis. Fixed cells were washed with PBS and stained by resuspension in PBS containing 2.5 μg/ml 7-AAD, 0.05% Triton X-100, 0.1 mg/ml RNAse A (Roche, 10109142001), followed by incubation for 30 minutes at 37 °C. Cells were not washed after this step and kept on ice until flow cytometry analysis. Flow cytometry was performed using the FACS Calibur flow cytometer (BD Biosciences). The cell cycle phase distribution analysis was performed with the software ModFit LT version 3.3 (Verity Software House) using the Sync Wizard with modeling of equally-spaced trapezoids for S-phase and debris and aggregate modeling.