We used the protocol previously described by Figueroa et al. (2010) (link). Briefly, 106 HL60 cells (IDH1 WT or R132H) was used for each condition. After wash with PBS, cells were stained with Zombie Aqua (#423101; Biolegend; 1/1,000e) for 15 min at room temperature, then washed with PBS. Cells were fixed and permeabilized with IntraPrep Permabilization Reagent (#A07803; Beckman Coulter) as described by the manufacturer. Cells were then treated with 2N HCL for 20 min at room temperature in the dark and subsequently neutralized with 100 mM Tris-HCl, pH 8.0, for 20 min. After wash with PBS, cells were incubated with anti–5-OH-methylcytosine (#39770, Active Motif; 1:400) or IgG Rabbit antibody (#2729S; Cell Signaling; 1:400) for 30 min at room temperature in the dark. After wash with PBS, cells were incubated with secondary antibodies coupled with ALEXA FLUOR 555 (#A21428; Invitrogen) for 30 min in the dark. Cells were then washed three times in PBS and analyzed with CytoFlex. Data analysis was done using CytExpert.