Orca flash4.0 v2 digital scmos camera

Cells were fixed in 4% paraformaldehyde in PBS for 15 min at RT, washed 3 times in PBS, and then blocked for 30 min at RT in 3% bovine serum albumin (BSA) and 0.1% Triton X-100 in PBS (PBSTx+BSA). Primary antibodies were pooled and diluted in PBSTx+BSA. Coverslips were incubated in primary antibodies for 1 h at RT and washed 3 times in PBSTx. Alexa Fluor (Invitrogen) secondary antibodies were pooled and diluted at 1:350 in PBSTx+BSA. Coverslips were incubated in secondary antibody for 45 min at RT and then washed twice with PBSTx. Coverslips were counterstained with DAPI and mounted on glass slides with Prolong Diamond anti-fade medium (Invitrogen) and allowed to cure overnight. Image acquisition was performed on a Nikon Eclipse Ti inverted microscope equipped with motorized stage, LED epifluorescence light source (Spectra X), 60x/1.4 NA (Plan Apo) objective, and ORCA Flash4.0 V2+ digital sCMOS camera (Hamamatsu).

For mitotic checkpoint experiments (Figure 2), AML cell lines stably expressing H2B-mScarlet were seeded onto poly-L-lysine coated glass-bottomed plates and grown to 90% confluency. Prior to imaging, cells were challenged with 0.2 μg/ml nocodazole for 4 h or left untreated. Image acquisition was performed on a Nikon Eclipse Ti inverted microscope equipped with motorized stage, LED epifluorescence light source (Spectra X), 20x/0.45 NA (Plan Apo) objective, and ORCA Flash4.0 V2+ digital sCMOS camera (Hamamatsu). Environmental control was maintained at 37 °C and 5% CO₂. Images were acquired every 2 min for 10 h. For Mps1 inhibitor experiments (Figure 3), K562 cells stably expressing H2B-mScarlet were seeded onto poly-L-lysine coated glass-bottomed plates and grown to 90% confluency. Prior to imaging, cells were challenged with 2 μM AZ3146 for 2 h or left untreated. Image acquisition was performed as described above. Images were acquired every 3 min for 24 h.