Making HT1080 metaphases

• grow cells in 10 ml DMEM/FCS/antibiotics in a medium (~75 cm²) flask to get an almost confluent cultur
• split the culture 1:2 and let grow overnight

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• add 100 µl colcemid (10µg/ml) to the 10 ml culture and continue to grow 1 - 2.5 hours
• the cells must get rounder, detach and change to a less translucent form (check under the microscope), if they have changed proceed to the next step
  • meanwhile: prewarm 75 mM (or 0.4% C.G.) KCl solution to 37°C
  • and: make fresh fixative (methanol/galcial acetic acid = 3/1) and chill at -20°C
• to remove mitotic cells shake the flask hard against a paper towel roll (well attached cells are probably not deviding)
• transfer the cells into a conical falcon tube
• wash the flask with a few ml PBS and transfer the PBS into the tube
• centrifuge 5 min 1000 rpm
• remove supernatant but leave about 1 ml to resuspend the cells
• wash again with 10 ml PBS
• centrifuge 5 min 1000 rpm
• remove supernatant but leave about 1 ml to resuspend the cells
• add slowly 10 - 40 ml prewarmed KCl solution
• resuspend the cells and incubate 12 min at 37°C
• centrifuge 5 min 1000 rpm
• remove supernatant but leave about 1 ml to resuspend the cells
• add slowly 10 ml fixative
• wash several times with fixative until no traces of salt remain when you drop the cells on a slide and evaporation is even
• store cells in fixative at -20°C