Axioimager m1 epifluorescence and brightfield microscope

WT and Trib2^-/- NH9 cells were seeded at 0.2 × 10⁶ cells/ml and treated with DNR/ DMSO from WT and Trib2^-/- NH9 cells after 24h DNR treatment from WT and Trib2^-/- NH9 cells after 24h DNR treatment for 24h. Demecolcine solution (Sigma) was added to the cell suspension (100 ng/ml) 45 min prior the end of the treatment. The cells were incubated at 37 °C for 15 min in 75 mM KCl (swelling). The cell were fixed in 3:1 methanol:acetic acid. Fixed cells were dropped on microscope slides and let dry at RT. Mounting medium with DAPI (Vector shield) was applied to each sample. The slides were examined at an Axio Imager M1 Epifluorescence and Brightfield Microscope (Zeiss) and pictures captured to show representative mitotic cells (Axio Vision software).

6 × 10⁴ cells per condition were incubated on poly-L-lysine coated Hendley-Essex 12 well glass microscope slides for 1 h before being fixed in 4% formaldehyde in PBS. The cells were permeabilized in 0.5% Triton-X-100 PBS followed by 2 h of blocking in 5% BSA, 0.2% Triton-X-100 TBS. Primary antibody was applied overnight in a humidified chamber at 4 °C. Appropriate secondary antibody (1:500 dilution) was applied for further 1 h incubation at room temperature after removal of a primary antibody using PBS 0.1% Tween 20. Antifade mountant with DAPI reagent (Thermo Fisher #P36962) was used to seal each sample and images were captured on the Zeiss Axioimager M1 Epifluorescence and Brightfield Microscope. CellProfiler v2.2.0 image analysis software (CellProfiler) was used to quantify IF signals.