Human Cell Line Maintenance

Materials: 25ml DMEM media, 10ml PBS, 2ml Trypsin, (5ml Pen/strep, 50ml FBS) 1 tissue culture plate, 1 15ml falcon tube.

If using fresh DMEM, defrost pen/strep and 50ml FBS in 37⁰C waterbath (1 hour before starting). Then add 50ml FBS and 5ml pen/strep to DMEM media – label with name and date. Warm DMEM, PBS and Trypsin in 37⁰C waterbath at least 30min in advance of starting.

1. Check cell confluency with microscope
2. Aspirate off old media (with vacuum pump
3. Wash cells with 10ml warm PBS and aspirate PBS off
4. Add 2ml Trypsin, move to incubator for 3-5min until cells begin to come off plate
5. Check cells have detached from plate (microscope)
6. Add 5ml DMEM media, wash plate thoroughly to re-suspend all cells, move to 15ml tube
7. Spin 4min at 1000rpm and room temp
8. Aspirate off media (do not disturb pellet)
9. Re-suspend cells in 1ml DMEM media, add a further 9ml DMEM. Mix thoroughly.
10. Plate 1ml culture and add 9ml DMEM (for a 1 in 10 dilution).
11. Swirl plate side to side and up and down (not in a circular motion)
12. Grow 2 days at 37⁰C and 5% CO2

Return DMEM and PBS to fridge, and pen/strep and trypsin to freezer.