Isotype rat igg1κ

Flow cytometric measurement of global methylation was performed as described previously (Delaney et al., 2012 (link)). Briefly, 2.5 × 105 freshly harvested T cells were stained with FITC anti-CD3 Abs and PE-Cy5 anti-CD4, or isotypes, then fixed in Cytofix/Cytoperm (BD) and permeabilized using PBS supplemented with 0.1% saponin, 1% FBS, and 0.1% sodium azide. Cells were treated with RNase A to eliminate the potential for the detection of 5-methylcytidine in tRNA. Cells were treated with anti-5-methylcytidine (Acris Antibodies, San Diego, CA, USA), washed, then incubated with Anti-mouse IgG1-PE or isotype rat-IgG1κ (BD). Flow cytometry was performed immediately or within 24 h of permeabilization using a FACScaliber machine (BD). Results were analyzed with FCS Express software (de novo Software, Los Angeles, CA, USA).