Passaging and Seeding Mouse Embryonic Fibroblasts (MEFs) for Experiments
madalynn.erb Erb
Abstract
This protocol details the passaging and seeding mouse embryonic fibroblasts (MEFs) for experiments.
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Steps
Passaging and Seeding Mouse Embryonic Fibroblasts (MEFs) for Experiments
Passage cells when confluency exceeds 60%.
Remove media.
Wash cells with 5mL prewarmed PBS without Calcium and magnesium.
Aspirate and discard PBS.
Add 3mL - 5mL Room temperature TrypLE Express (enough to ensure complete coverage of cells).
Incubate cells at 37°C until they have detached.
Check at 0h 3m 0s - 0h 5m 0s intervals under an inverted microscope, gently tap flask to dislodge cells if needed.
Add 5mL - 10mL (or at least 1:1 ratio of media : TrypLE) of pre-warmed complete media to flask.
Pipette up and down to dislodge cells as needed.
Transfer cell suspension to conical tube.
Spin down at 1300rpm,0h 0m 0s - 1800rpm,0h 0m 0s (300x g,0h 0m 0s) for 3 mins - 0h 5m 0s.
During this time label one microcentrifuge tube for each cell line being used and add 20µL of Trypan blue.
Check for cell pellet then aspirate and discard supernatant.
Avoid disturbing the cell pellet.
Resuspend cells in appropriate volume of prewarmed complete media.
Take 20µL of resuspended cell mix (gently pipetting up and down 10 times) and add to 20µL of trypan blue.
Pipette up and down 10 times to mix cells with trypan blue.
Count the cells using a Countess 3 (Thermofisher Scientific).
Clean the glass hemocytometer slide and add 10µL of cell / trypan blue mix to chamber A and B.
Avoid bubbles and dirt.
Perform cells count ensuring working in same units.
Once counts are performed mix cell suspension again as cells will have settled to the bottom of the tube by the time counts are done.
Add ______________ μL/mL of to ___________ μL/mL of media
Seed cells in appropriate volumes.
Record the following:
- Cell type, strain and genotype*
- Number of wells
- Density per well*
- Passage*
- Time and Date of Seeding*
- Temperature(if not at standard room temp)
If left over cell are needed, place in an appropriate volume of media and transfer to a fresh flask for future experiments
Incubate plate and flasks 0h 5m 0s (O/N) in 37°C incubator.
