Alexa 647 conjugated anti rabbit igg

Cell pellets were fixed and permeabilized with dropwise addition of 1 ml of cold 70% ethanol and then incubated overnight. All steps were at 4°C or on ice. Samples were then washed, blocked and incubated for one hour in flow buffer (PBS with 2% FBS and 2 mM EDTA) with each of the following antibodies; Alexa 647 conjugated rabbit anti histone H3 tri-methylated lysine 27 diluted 1:50 (Cell Signaling Technology #12158), unconjugated rabbit polyclonal anti histone H3 tri-methylated lysine 9 (Abcam #8898) , or APC conjugated rabbit anti-human Ki-67 antibody (Biolegend #350513). Cells were washed twice with flow buffer. The unconjugated histone H3 tri-methylated lysine 9 antibody was detected with an Alexa 647 conjugated anti-rabbit IgG diluted 1:250 (Cell Signaling Technology #4414). Data was acquired with a three laser (405 nm, 488 nm and 640 nm) Becton Dickinson FACS Aria IIu flow cytometer. Gating was forward scatter vs side scatter, single cells (linear on FSC-A vs FSC-H), then the 670/30 filter (APC or Alexa 647) vs forward scatter or histogram. An isotype control antibody was used for setting the gates. Negative and dim cells were selected for methylated histones. Negative cells were selected for Ki67. Data is presented as representative plots or mean ± SEM of triplicate wells from replicate experiments.