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Team:Warwick/Notebook/cellmaintenance
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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. | 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. | Warm DMEM, PBS and Trypsin in 37⁰C waterbath at least 30min in advance of starting. | ||
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Latest revision as of 10:21, 27 August 2014
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.
- Check cell confluency with microscope
- Aspirate off old media (with vacuum pump
- Wash cells with 10ml warm PBS and aspirate PBS off
- Add 2ml Trypsin, move to incubator for 3-5min until cells begin to come off plate
- Check cells have detached from plate (microscope)
- Add 5ml DMEM media, wash plate thoroughly to re-suspend all cells, move to 15ml tube
- Spin 4min at 1000rpm and room temp
- Aspirate off media (do not disturb pellet)
- Re-suspend cells in 1ml DMEM media, add a further 9ml DMEM. Mix thoroughly.
- Plate 1ml culture and add 9ml DMEM (for a 1 in 10 dilution).
- Swirl plate side to side and up and down (not in a circular motion)
- Grow 2 days at 37⁰C and 5% CO2
Return DMEM and PBS to fridge, and pen/strep and trypsin to freezer.
