Cultivation of HEK293T cells

Materials

Medium: DMEM + 10 % calf serum (CS), PBS, Trypsin / EDTA

Procedure:

• Remove growth medium with sterile pasteur pipette from dishes

• Wash 1x carefully with PBS, remove PBS

• 1 ml Trypsin/EDTA on cells, short incubation (max 2 min)
• If cells are detached, stop activity of trypsin with addition of 4 ml medium
• Transfer cell solution in falcon tube, centrifugation 800rpm, 3 min, room temperature
• Remove medium/ trypsin mixture, add new medium and mix
• Optional - For assays with specific cell numbers:
  • count cells in Neubauer counting chamber or use CasyTT: add 25 μl of cell solution in Casy tube
  • add 5 ml of Casy Ton
  • measure cell number
• For normal cultivation (two days) or transfection at next day: divide cells 1:5 in new dishes; for cultivation over weekend (three days): divide cells 1:10 in new dishes