Transcript MEF Medium - Bio-Link

Tuesday Lab
Make media and plate MEFs
Cell Culture: Best Practices
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PLAN AHEAD and FOCUS
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Avoid CONTAMINATION
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assemble tubes, pipettes, & reagents BEFORE you start
Thaw enough feeder cells beforehand
Focus on one activity at a time
Clean all items brought into the hood with EtOH
Watch to see if pipette tip has touched a potentially contaminated surface
Ensure liquids don’t get sucked into pipette tools
Immediately clean any spilled media or cell suspension
GOOD TECHNIQUE
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Pipette gently to avoid creating bubbles or aerosols
Don’t use circular motions to spread freshly plated cells—rock back and forth
Mix reagents thoroughly after thawing
Clear biosafety cabinet vents
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Making MEF Medium
MEF Medium (250ml)
225 ml DMEM with Glutamax
25 ml FBS (10%)
2.5 ml Pen/Strep
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Making Differentiation Medium
Differentiation Media
DMEM with Glutamax
FBS
Pen/Strep
250 ml
200 ml
50 ml
2.5 ml
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hESC/hiPSC Medium
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
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Thawing MEFs:
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[~30’ 37C]
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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).
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Thawing MEFs (1):
• Prepare MEF dishes (day1)
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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.
Thawing MEFs (2):
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.
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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.
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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)
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