TITLE: Passaging and Seeding Mouse Embryonic Fibroblasts (MEFs) for Experiments
AUTHORS: madalynn.erb

[DESCRIPTION]
This protocol details the passaging and seeding mouse embryonic fibroblasts (MEFs) for experiments.

[STEPS]
SECTION: Passaging and Seeding Mouse Embryonic Fibroblasts (MEFs) for Experiments
1. Passage cells when confluency exceeds 60%.
SECTION: Passaging and Seeding Mouse Embryonic Fibroblasts (MEFs) for Experiments
2. Remove media.
SECTION: Passaging and Seeding Mouse Embryonic Fibroblasts (MEFs) for Experiments
3. Wash cells with 5 mL prewarmed PBS without Calcium and magnesium.
SECTION: Passaging and Seeding Mouse Embryonic Fibroblasts (MEFs) for Experiments
4. Aspirate and discard PBS.
SECTION: Passaging and Seeding Mouse Embryonic Fibroblasts (MEFs) for Experiments
5. Add 3 mL - 5 mL Room temperature TrypLE Express (enough to ensure complete coverage of cells).

SECTION: Passaging and Seeding Mouse Embryonic Fibroblasts (MEFs) for Experiments
6. Incubate cells at 37 °C until they have detached.
SECTION: Passaging and Seeding Mouse Embryonic Fibroblasts (MEFs) for Experiments
6.1. Check at 3 min - 5 min intervals under an inverted microscope, gently tap flask to dislodge cells if needed.

SECTION: Passaging and Seeding Mouse Embryonic Fibroblasts (MEFs) for Experiments
7. Add 5 mL - 10 mL (or at least 1:1 ratio of media : TrypLE) of pre-warmed complete media to flask.

SECTION: Passaging and Seeding Mouse Embryonic Fibroblasts (MEFs) for Experiments
7.1. Pipette up and down to dislodge cells as needed.
SECTION: Passaging and Seeding Mouse Embryonic Fibroblasts (MEFs) for Experiments
8. Transfer cell suspension to conical tube.
SECTION: Passaging and Seeding Mouse Embryonic Fibroblasts (MEFs) for Experiments
9. Spin down at 1300 rpm - 1800 rpm (300 x g) for 3 mins - 5 min.

SECTION: Passaging and Seeding Mouse Embryonic Fibroblasts (MEFs) for Experiments
9.1. During this time label one microcentrifuge tube for each cell line being used and add 20 µL of Trypan blue.
SECTION: Passaging and Seeding Mouse Embryonic Fibroblasts (MEFs) for Experiments
10. Check for cell pellet then aspirate and discard supernatant.
SECTION: Passaging and Seeding Mouse Embryonic Fibroblasts (MEFs) for Experiments
10.1. Avoid disturbing the cell pellet.
SECTION: Passaging and Seeding Mouse Embryonic Fibroblasts (MEFs) for Experiments
11. Resuspend cells in appropriate volume of prewarmed complete media.
SECTION: Passaging and Seeding Mouse Embryonic Fibroblasts (MEFs) for Experiments
12. Take 20 µL of resuspended cell mix (gently pipetting up and down 10 times) and add to 20 µL of trypan blue.
SECTION: Passaging and Seeding Mouse Embryonic Fibroblasts (MEFs) for Experiments
12.1. Pipette up and down 10 times to mix cells with trypan blue.
SECTION: Passaging and Seeding Mouse Embryonic Fibroblasts (MEFs) for Experiments
13. Count the cells using a Countess 3 (Thermofisher Scientific).
SECTION: Passaging and Seeding Mouse Embryonic Fibroblasts (MEFs) for Experiments
13.1. Clean the glass hemocytometer slide and add 10 µL of cell / trypan blue mix to chamber A and B.
SECTION: Passaging and Seeding Mouse Embryonic Fibroblasts (MEFs) for Experiments
13.2. Avoid bubbles and dirt.
SECTION: Passaging and Seeding Mouse Embryonic Fibroblasts (MEFs) for Experiments
14. Perform cells count ensuring working in same units.
 
A=________	B=________	Average A & B =_____________
SECTION: Passaging and Seeding Mouse Embryonic Fibroblasts (MEFs) for Experiments
15. 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
SECTION: Passaging and Seeding Mouse Embryonic Fibroblasts (MEFs) for Experiments
16. Seed cells in appropriate volumes.
SECTION: Passaging and Seeding Mouse Embryonic Fibroblasts (MEFs) for Experiments
17. 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)
SECTION: Passaging and Seeding Mouse Embryonic Fibroblasts (MEFs) for Experiments
18. If left over cell are needed, place in an appropriate volume of media and transfer to a fresh flask for future experiments
SECTION: Passaging and Seeding Mouse Embryonic Fibroblasts (MEFs) for Experiments
19. Incubate plate and flasks 5 min (O/N) in 37 °C incubator.