1. Tuesday Lab Make media and plate MEFs

2. Cell Culture: Best Practices 2 PLAN AHEAD and FOCUS 1.assemble tubes, pipettes, & reagents BEFORE you start 2.Thaw enough feeder cells beforehand 3.Focus on one activity at a time Avoid CONTAMINATION 1.Clean all items brought into the hood with EtOH 2.Watch to see if pipette tip has touched a potentially contaminated surface 3.Ensure liquids don’t get sucked into pipette tools 4.Immediately clean any spilled media or cell suspension GOOD TECHNIQUE 1.Pipette gently to avoid creating bubbles or aerosols 2.Don’t use circular motions to spread freshly plated cells—rock back and forth 3.Mix reagents thoroughly after thawing 4.Clear biosafety cabinet vents

3. Making MEF Medium 3 MEF Medium (250ml) 225 ml DMEM with Glutamax 25 ml FBS (10%) 2.5 ml Pen/Strep

4. Making Differentiation Medium 4 Differentiation Media 250 ml DMEM with Glutamax 200 ml FBS 50 ml Pen/Strep 2.5 ml

5. hESC/hiPSC Medium 5 hESC Medium 250 ml (W8 media) Dmem/F12 Knockout media 196ml Knock Out Serum Replacer (KOSR) 50ml Non-essential Amino Acids (NEAA)2.5ml Glutamine 200x 2.5ml Pen/Strep 2.5ml 55nM Beta –Mercaptoethanol 455ul FGF 10ug/ml 200ul

6. Thawing MEFs: 1 Add 1 ml gelatin solution to 24 [20] 6-wells (for mESC [hESC] culture) and keep in tissue culture incubator for 30’. 2 Prepare a 50 ml falcon tube with 5 ml warm MEF medium. 3 Take frozen vial with MEFs, hold in 37C water bath (hold by its lid - do not let lid get wet!) until a small piece of ice remains, then quickly ethanol-spray and wipe, move vial to the hood, transfer a small amount of warm media from the falcon tube to the MEF tube, slowly mix by pipetting up and down for a few times. 4 Transfer resuspended MEFs to the falcon tube (pipette slowly). 5 Spin tube. 6 Aspirate supernatant above the cell pellet and 7 the gelatin from the wells. 8 Resuspend MEFs in 5 ml of warm MEF media (pipette several times up and down), then adjust to volume to the desired amount =24 ml [20 ml] for MEFs for mESC [hESC] culture. 9 Plate MEFs (2 ml per well) 10 Transfer the plates into the incubator, then rock plate back/forth and left/right (avoid circular motions!) a few times to ensure cells settle evenly within the dish or well(s). 3 4 1 2 [~30’ 37C] 7 6 8 10 6 9 5

7. Thawing MEFs (1): Prepare MEF dishes (day1) 1. Estimate Number of dishes needed-- MEFs are normally frozen down in convenient of numbers of cells, for example for 4 dishes or 2 6-well plates. 2.Pre-coat dishes with 0.25% gelatin solution. Use just enough to cover bottom of plate. Incubate at least for 30 minutes at room temperature. In a 15 ml vial add 10 mls of MEF media. 3.Rapidly thaw vials of MEFs in 37 C. water bath until almost completely thawed(there should still be some ice crystals still in vial). Spray vial with ethanol and wipe dry and move to hood. 4.Open vial and add small amount of MEF media to cryovial and then transfer MEFs to 10 ml vial with media.

8. Thawing MEFs (2): 8 1.Centrifuge 5 minutes at 1000 rpms. 2.Return to hood after spraying with ethanol. Aspirate the media and re-suspend to appropriate volume of MEF media. Remove the gelatin from the dish and immediately add the MEFs to the dish without letting the gelatin dry. 3.Incubate the MEFs overnight. 4. On the next morning, observe the settled feeder monolayer under a high quality microscope and make sure feeder layer is confluent. The entire dish should be covered with cells.

9. Thawing MEFs: Five Important Things to Remember when Thawing Mouse Embryonic Feeder Cells Hold frozen vial with MEFs, in 37C water bath by its lid - do not let lid get wet! When transferring warm media from the falcon tube to the MEF tube, slowly mix by pipetting up down for a few times. Pipette slowly when transferring re-suspended MEFs to the falcon tube. Re-suspend MEFs in 3 ml of warm MEF media by pipettimg several times up and down. then adjust to volume to the desired amount. Avoid circular motions! Transfer the plates into the incubator, then rock plate back/forth and left/right a few times to ensure cells settle evenly within the dish or well(s). (IMAGE) 9