Software vx 0

DLN single-cell suspensions were generated by mechanical disruption and passed through a 70 μm cell strainer. Except for cell cycle staining, cell suspensions were labelled with the Fixable Viability Dye eFluor 450 (eBioscience, Carlsbad, CA) to exclude dead cells from the analysis. Cell suspensions were blocked with universal blocking reagent Power Block (BioGenex, CA, USA) and incubated with the relevant anti-mouse antibodies (detailed in supplemental Table 1). After two washes, cell suspensions were treated for intracellular staining with BD Cytofix/Cytoperm (San Jose, CA) according to manufacturer specifications and further incubated with anti-IgM FITC and anti-IgG APC or anti-active-Caspase3 FITC antibodies. For cell cycle analysis, after surface staining, cell suspensions were treated with the Foxp3/Transcription Factor Fixation/Permeabilization Foxp3 kit (eBioscience, Carlsbad, CA) for intracellular/intranuclear detection of Ki-67. Cells were washed once and then incubated 10 min with Hoechst 33258 (Polysciences, Warrington, PA) for DNA labelling.
Finally, cells were fixed with 1% paraformaldehyde (Sigma-Aldrich, St. Louis, MO) and acquired in a CytoFLEX LX flow cytometer using CytExpert software (Beckman Coulter, Indianapolis, IN). Data was analyzed using FlowJo software vX.0.7 (Ashland, OR).

Single-cell suspensions were surface-stained with fluorescently conjugated Abs against murine PerCP-Cy5.5-conjugated CD4 (RM4-5), BV510-conjugated CD8 (53-6.7), BV510-conjugated CD8 (53-6.7), PE-conjugated CD8 (53-6.7), FITC-conjugated CD44 (IM7), APC-conjugated CD62L (MEL-14), or APC-conjugated CD25 (PC61) (BD Biosciences, NJ, USA).
For intracellular cytokine staining (ICS) of BV421-conjugated IFN-γ (XMG1.2) (BD Biosciences), activated T cells were stimulated with leukocyte activation cocktail (BD Biosciences) containing PMA, ionomycin, and the protein transport inhibitor BD GolgiPlug™ (Brefeldin A) for 4 hours, then were first stained for surface markers, followed by ICS with a Cytofix/Cytoperm kit (BD Biosciences).
For transcription factor (TF) staining of PE-conjugated Foxp3 (MF23) (BD Biosciences), activated T cells were first stained for surface markers, then stained with a TF Fix/Perm kit (BD Biosciences).
Flow cytometry was performed on FACSCelesta and LSRFortessa cytometers (BD Biosciences). Analysis was performed with FlowJo software (vX.0.7).