Anti prb pe conjugated clone g3 245

U937 were washed in PBS for 10 min at RT, resuspended in 300 μL formaldehyde 3.7% and incubated at 4 °C for 15 min; 2 mL perm/washing buffer was added and cells were centrifuged at 1200 rpm for 10 min. Pellets were resuspended in 300 μL Cytoperm reagent [101 (link)]. Monoclonal anti-human antibodies Anti-pRb PE conjugated (clone G3-245) (BD) and anti bcl-2 FITC (clone 124) (Dako, Milan, Italy) were added to samples at concentrations according to the manufacturer’s instructions. Cells were incubated at 4 °C for 30 min before being processed by flow cytometry and/or confocal microscopy.

The intracellular content of molecules was revealed by previous cell fixation and permeabilisation (FIX & PERM^® Cell Fixation and Cell Permeabilisation Kit; Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA). Cells were fixed and permeabilised, then the fixed cells were stained with monoclonal anti-human antibodies anti-pRb PE-conjugated (clone G3-245) (BD Bioscience, Franklin Lakes, NJ, USA) and RelA/NFkB p65 Alexa Fluor 488-conjugated (112A1021, Novus Biologicals, Littleton, CO, USA) was added to samples at concentrations according to the manufacturer’s instructions. Proliferating cell nuclear antigen expression was studied in cells fixed in 70% ethanol using anti-PCNA FITC-conjugated (clone P10, Sigma-Aldrich, St. Louis, MO, USA).