Monitoring mitotic cell division in DT40 cells - Events

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Method Electroporation of DT40 cells (II) • Use 5 x 10 6 cells. Cells must be in the log growth phase and I always split them approx 1:4 the day before electroporation • On the day of electroporation count cells and spin down 5 x 10 6 cells. Resuspend cells in 1ml of optimem + 1% DMSO. Add plasmid DNA and put the cells + DNA into an electroporation cuvette • Electroporate at 300 volts, 960μF • Dilute cells into 20 ml DT40 growth medium and incubate O/N at 39.5 o C, 5% CO 2
Isolation of DT40 clones • The next day, take the volume up to 140 ml with DT40 growth medium containing the required selection agent (e.g. G418 at 1.5 mg/ ml) and 50 µg/ ml amphotericin (fungicide) • Split the 140 ml between 7 x 96 well plates using a multi-channel pipette (200 μl per well). Incubate at 39.5 o C, 5% CO 2 • After approx 3 weeks examine the wells for small colonies (balls) of antibiotic resistant cells. Clones expressing H2B:GFP exhibit green fluorescence and are easily identified! • Transfer a few clones into 24 well plates and progressively expand them • Verify DNA fluorescence and that the stages of mitosis look OK by confocal microscopy. Choose the appropriate clone(s)
Page 1 and 2: Tubulin DNA Monitoring mitotic cell
Page 3 and 4: Time-lapse video microscopy • Cel
Page 5 and 6: Components of a time-lapse video mi
Page 7 and 8: Components of a time-lapse video mi
Page 9 and 10: Generation of DT40 cells stably exp
Page 11: Electroporation of DT40 cells (I) I
Page 15 and 16: Set up cells Setting up DT40s for t
Page 17 and 18: Monitoring mitotic cell division by
Page 19 and 20: metaphase anaphase Normal chromosom
Page 21 and 22: Chk1-deficient Chk1 deficient DT40
Page 23: Living cell Apoptotic cell Mitotic

Monitoring mitotic cell division in DT40 cells - Events

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