Pfaff Lab Feeder Preparation/Maintenance Responsibilities and Protocols

    Order live/treated PMEFs from Chemicon and store at -80°C:
  1. Make sure we always have some in the lab and order more as they get low
  2. Neo resistant  catalog # PMEF-NL
  3. Hygro resistant  catalog # PMEF-HL
  4. Order some that are already mitomycinC-treated as backup
  5. #PMEF-N
  6. #PMEF-H
    Expand live PMEFs:
  1. PMEFs come at passage 3 (expand to passage 5)
  2. Thaw 2 vials onto two large dishes pre treated with gelatin in PMEF media (25 ml):
    1. PMEF media (prepare 1 liter):
    2. Prepare dishes by adding 15 ml of 0.1% gelatin for 15 minutes
    3. Add 1 ml from vial to 2 ml of PMEF media in 15 ml conical
    4. Spin 100 rpm for two minutes
    5. Resuspend in 5 ml media
    6. Add 5 ml to plate with 20 ml PMEF media (total of 25 ml)
  3. After 3-4 days (when cells are very confluent), split PMEFs 1:3:
    1. Prepare dishes by adding 15 ml of 0.1% gelatin
    2. Aspirate media
    3. Wash with PBS
    4. Trypsinize with 5 ml 0.25% trypsin
    5. Add 5 ml PMEF media and resuspend
    6. Pellet at 1000 rpm for 5 minutes
    7. Resuspend in 9 ml of PMEF media
    8. Aliquot 3 ml onto 3 fresh plates (pre-treated with gelatin) that have 22 ml fresh PMEF media
    9. Start with two plates-should now have 6 plates
  4. After 3-4 days (when cells are very confluent), split PMEFs 1:3:
    1. Same protocol as above
    2. 6 plates onto 18 plates
    Growth arrest and freeze PMEFs:
  1. In the morning, treat PMEFs with mitomycinC (wear gloves!!!):
    1. Resuspend 2 vials of mitomycinC in 20 ml of PMEF media
    2. Add 1ml of media to each vial
    3. Pipette up and down and mix thoroughly
    4. Add each to 18 ml of media
    5. Rinse remaining vial with media again to get most of it out
    6. Warm to 37°C to help it into solution
  2. Add 1ml mitomycin C to each plate for 3 hours
  3. Harvest PMEFs:
    1. Prepare freezing medium:
    2. Aspirate media from dishes
    3. Wash with PBS
    4. Trypsinize and collect cells as described above
    5. Pull pellets into 50 ml conical tube
    6. Resuspend in 50 ml of freezing medium
    7. Aliquot 1ml into cryotubes and place in styrofoam and place in -80°C
    8. Next day move into labeled freezer box
    Test Feeder density/survival:
  1. Thaw 1 vial of PMEFs and plate onto 1 10 cm dish:
    1. Gelatinze dish with 7 ml gelatin
    2. Rapidly thaw vial at 37°C
    3. Add 1 ml of cells to 15 ml concial tube with 2 ml of PMEF media
    4. Spin 1000 rpm 2 minutes
    5. Resuspend in 10 ml medium and add to gelatinized dish
  2. Observe feeders for cell density and note in notebook (1x  1.5x  2x)
    Thaw feeders as requested on feeder log sheet:
  1. 10 cm dish = one 6 well dish = one 12 well dish = one 24 well dish = one 96 well dish
  2. Gelatinize dishes depending upon how many are requested that day
  3. Prewarm media (need 12 ml per dish + 2 ml wash per vial = 14 ml)
  4. Rapidly thaw vial at 37°C
  5. Add 1 ml cells to 2 ml PMEF media
  6. Spin 1000 rpm for 2 minutes
  7. Resuspend and plate accordingly

Example:

One 6 well dish:
Thaw one 1X vial
Add 1 ml cells to 2 ml media
Spin 1000 rpm 2 minutes
Resuspend in 12 mls
Aliquot 2 ml to each well


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