Igg rabbit antibody

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; 1/1,000^e) 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.