MEM-α media was removed from transfected cells on poly-D-lysine–coated filming dishes (MatTek) and replaced with CO2-independent imaging media (Gibco) supplemented with 10% FBS and 1% penicillin and streptomycin. Mitotic cells were identified by scanning for GFP-CENP-B fluorescence using the 60× objective and imaged with the 100× objective using 0.5-µm z-stacks every 2 s for ∼5 min. After imaging, the XY position of the cell was marked and revisited within an hour of imaging to determine if the cell had divided for quality control. For mCherry- or mCherry-KBP–expressing cells, 0.5-µm z-stacks of the entire cell were taken in TRITC and GFP channels before time course imaging of GFP-CENP-B.
Poly d lysine coated filming dishes
Manufactured by MatTek
Poly-D-lysine–coated filming dishes are a type of cell culture dish designed to promote cell attachment and growth. The dishes are coated with the synthetic polymer poly-D-lysine, which enhances the adhesion of various cell types to the dish surface.
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2 protocols using poly d lysine coated filming dishes
1
Live-cell Imaging of Mitotic Cells
2
Live-cell Imaging of Mitosis
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Culturing medium was removed from transfected cells plated on poly-d -lysine–coated filming dishes (MatTek) and replaced with CO2-independent imaging medium (Gibco) supplemented with 10% FBS and 1% penicillin and streptomycin. Using the 40× objective with differential interference contrast (DIC) and GFP fluorescent imaging, 10 randomly selected XY fields were programmed for imaging every 2 min for 16 h. For analysis, nuclear envelope breakdown and anaphase onset time points were determined for cells that completely divided and were detectably expressing GFP.
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