Facscaliber machine

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).

PBMCs and CD4+ cells infected with NL4-3wt or NL-AD8 were strained with anti-p24 FITC-conjugated or PE-conjugated antibody (KC57, Beckman Coulter) using the Cytofix/Cytoperm and the Perm/Wash buffers (BD Biosciences) according to manufacturer’s instructions. Cells were acquired with a FACSAriaIII or FACSCaliber machine (BD Biosciences) using 488 and 640nm laser lines. A minimum of 10⁵ cells per sample were acquired. Results were analyzed with FlowJo 10.0.8 software. For CFP/YFP co-infection experiments, cells were acquired with a FACSAriaIII using the 405nm laser line for CFP, and 488nm laser line for YFP.