Imaging Flow Cytometry

B16-F10 cells treated with Navtemadlin or DMSO control, were harvested and single-cell suspensions were incubated with Fc Block (clone 2.4G2, BD Biosciences) for 10 minutes at room temperature to reduce unspecific antibody binding. Intracellular staining to detect p53, p21, Bax, PUMA, BCL-2, and MCL-1 Ps159 was performed using the FoxP3 staining kit (Thermo Fisher Scientific) according to the manufacturer's instructions. Briefly, cells were fixed in fixation/permeabilization buffer for 30 minutes at 4°C and washed with permeabilization buffer. Fixed cells were then incubated for 30 minutes at 4°C with antibodies against intracellular proteins: p53-Alexa 647, 1:75 (1C12, Cell Signaling Technology); p21-Alexa 488, 1:100 (F-5);Bax-Alexa 595, 1:100 (B-9); PUMA-PE, 1:100 (B-6) from Santa Cruz Biotechnology; BCL-2-PE-Vio770, 1:10 (REA356, Miltenyi Biotec) and MCL-1 Ps159-PE, 1:50 (REA924, Miltenyi Biotec).

After washing, single-cell suspensions were resuspended in flow buffer (0.5% BSA, 2 μmol/L EDTA in PBS) containing the DNA stain FxCycle Violet (1:2,000 dilution, Thermo Fisher Scientific). A minimum of 30,000 cells were acquired on an ImageStreamX Mk II Imaging Flow Cytometer (Amnis corporation) equipped with 405, 488, 561, 642, and 785 nm lasers at 60 × magnification. Data analyses were performed using IDEAS Software (Amnis Corporation).