Cultivation of HEK293T cells: Difference between revisions
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Created page with "'''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..." |
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'''Materials''' | == '''Materials''' == | ||
Medium: DMEM + 10 % calf serum (CS), PBS, Trypsin / EDTA | Medium: DMEM + 10 % calf serum (CS), PBS, Trypsin / EDTA | ||
'''Procedure:''' | == '''Procedure:''' == | ||
* Remove growth medium with sterile pasteur pipette from dishes | * Remove growth medium with sterile pasteur pipette from dishes | ||
| Line 18: | Line 16: | ||
** measure cell number | ** 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 | * 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 | ||
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Latest revision as of 15:45, 29 May 2025
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
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