Materials:
· cell culture media
VICs: M199 + 75 ml FBS + 5 ml Pen-Strep + 1 ml gentamicin + 5 ml L-glutamine
3T3s: DMEM + 50 ml FBS or Calf Serum + 5 ml Pen-Strep
· phosphate buffered saline (PBS)
· trypsin/EDTA
Preparation:
· warm media, PBS, and trypsin to 37°C in water bath
· wipe laminar flow hood down with 70% ethanol
· wipe all containers that enter hood with 70% ethanol
Procedure: (for 10 mm dishes)
1. Aspirate old media from dish.
2. Add 8 ml PBS to dish. Always tip dish to the side when adding solutions so as not to shear the cells attached to the bottom surface.
3. Aspirate off PBS.
4. Add 5 ml of trypsin. Tilt dish to ensure that the entire bottom surface is covered with solution.
5. Place in 37°C incubator for 2-6 minutes.
6. Remove dish from incubator and gently tap it to loosen the cells. Inspect dish either macroscopically or microscopically to ensure that all cells have been cleaved from the surface. If not, either tap dish a few more times or return to incubator for 1-2 minutes longer.
7. Add 4 ml of media to the dish.
8. Remove all contents of the flask and place in 15 ml centrifuge tube.
9. Rinse dish surface with 4 ml PBS or 4 ml media and add to same centrifuge tube.
10. Tightly cap centrifuge tube, make appropriate counterweight, and centrifuge at 2000 rpm for 5 minutes (3T3s) or 1000 rpm for 6 minutes (VICs).
11. While the cells are centrifuging, prepare new dishes for cells to be passaged into. Add 8-10 ml media to each dish and label appropriately.
12. There will be a visible cell pellet at the bottom of the tube following centrifugation. Aspirate off as much supernatant as possible without disturbing the cells.
13. Resuspend cells in media (normally 5-6 ml). If seeding cells for an experiment, remove 10 ml and perform a hemacytometer count or coulter count.
14. Add the appropriate amount of cell suspension to each dish (1:3 is the recommended passage ratio for VICs; 1:50 is good for 3T3s) and place dishes in the incubator.
Clean-up
· All materials that have touched cells or media must be thrown in biohazard waste.
· Materials that have only touched trypsin, buffer, etc., can be thrown in regular trash.
· Wipe down hood with 70% ethanol.
· Turn on UV light, turn off blower, and close sash.
I. Seeding MEF cells
1) Take the cells from the minus 80
2) Leave them in the bath until you see a small ice ball; don’t leave them completely melt
3) Slowly take the cells with the Pasteur Pipette
4) Put the cells in a 15 mL Falcon tube
5) Add 5mL of MEF Medium drop by drop
6) Spin the tube at 1000tr/min for 5 minutes
7) Take a flask, add 15mL of MEF medium, write the date and P0 (passage0)
8) Take the tube and remove the medium and resuspend the cells with 2mL of medium
9) Seed the cells in the flask and leave them in the incubator.
Preparing mitomycin:
- 250 mL of DMEM
- 2mg of mitomycin
II. Passage MEF with mitomycin
1) Remove the media that is in the flask
2) Add the entire tube of motomycin (about 6 to 7 mL)
3) Leave the flask in the incubator for 2 hours
4) After 2 hours, remove all the mitomycin
5) Wash 4 times with PBS
6) Remove the last PBS washing
7) Add 2mL of trypsin
8) Put the flask in the incubator for about 2 minutes
9) Take the flask from the incubator and add 5 to 6 mL of MEF Medio to stop the trypsin
10) Pipette up & down around 10 times and and put in a 15 mL falcon tube
11) Spin down at 1400 for 5 minutes
12) During that time, take plates from the incubator, remove the gelatin and add MEF Medio
13) Resuspend the cells and put them in the plates : usually 1 flask into 4 10cm plates
1) Take out the media from the plates
2) Add 4mL of collagenase in each plate
3) Leave the flask in the incubator for 30 to 45 minutes
4) Add 5 mL of ES media
5) Wash the plategently to remove the ES colonies from the plate
6) Put them in a 15mL falcon tube
7) Wash a second time with ES media
8) Spin down at 700 for about 3 minutes
9) During that time, take new plates from the incubator, remove the gelatin and add 10mL of ES Medio
10) Resuspend the pellet by pipet up & down strongly
11) Spin down at 700 for about 3 minutes
12) Up& down strongly
13) Resuspend the cells and add to the plates
14) Put the plates in the incubator
For EB’s, put EB medium in a petry dish
Use Colleganse type 4 from Gibco – Invitrogen
1 mg collegenase /mL of DMEM without serum and then filtuer with 0.2 um filter and only use for 2 weeks.
1) Take out the media from the plates
2) Wash one time with PBS
3) Add 3mL of trysin (0.25 or 0.1)
4) Leave the flask in the incubator 5 minutes
5) Add 6 mL of TNS
6) Up & down many times to remove all the cells
7) Put them in a 15mL falcon tube
8) Pipet strongly
9) Take a Pasteur pipet and remove the gelatin
10) Spin down at 700 for about 3 minutes
11) During that time, take new plates from the incubator, remove the gelatin and add 10mL of ES Medio
12) Resuspend the cells and add to the plates (usually split 1 to 4)
13) Put the plates in the incubator
For EB’s, put in EB medium in petry dish