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Aurora Burds Connor, Alison Hayward, John Mkandawire, and Peimin Qi — Nov 2006
To increase the chance of generating numerous high percentage chimeras, it is very important to have non-stressed, healthy ES cells that are free of feeders (MEFs) and cell debris. This protocol provides for both by allowing cells to spend a few days recovering from the stress of freezing and by selectively enriching for ES cells. If cells are thawed closer to the injection date, they are visibly less healthy and more difficult to inject.
Our recipe for ES cell media is listed at the bottom of this protocol.
All our stocks of targeted ES cells are prepared such that 1 vial of cells can be thawed to one well of a 24-well plate. (1/3 to 1/4 of the cells in a confluent 24-well was frozen in a vial for this purpose.) If your stocks have fewer cells (from a 96 or 48-well plate), then you will need to begin your preparations much earlier.
Plate feeders on a gelatinized 24-well plate (about 7.5x104 cells/each 24-well, or 1.8x106 cells for an entire 24well plate.)
Around noon, thaw a vial of ES cells (1/3 to 1/4 of a confluent 24-well is previously frozen as stock for this), spin through 10mL media to remove the DMSO, pipette 10 times to resuspend in 500uL media into a 24-well.
Alternately, if you have a 48-well of your ES cells growing well, they can be split into a 24-well now. A 96-well to 24-well tends to be too great a split and the cells are less happy.
Feed the 24-well first thing in the morning. Coat a 6-well plate with ES-grade gelatin, then plate ½ the normal number of feeders onto this (about 2x105 cells/each 6-well)
In the morning, feed the 24 well, , wait 2-4 hours, then trypsinize (200uL 0.25% trypsin, NOT 0.05%), spin through 5mL media (low speed, about 125g, 3min), resuspend in 2mL and transfer to the 6-well with feeders.
If your 24-well of ES cells was VERY confluent, you can freeze back 1/4 of these cells to one stock vial, transferring the remaining 3/4 of the cells to the 6-well.
Feed the 6 well first thing in the morning.
Because your cells are on sparce feeders, some will have a flat, black and often spiky appearance. You may see a few glowing colonies as usual, but do not be alarmed by a morphology that is similar to differentiation. Feed the cells again before you leave for the day.
Warm the media, PBS, and trypsin.
Feed the cells, coat a new 6-well plate with gelatin.
Rinse cells in 2mL PBS, then add 600uL trypsin (again, 0.25% trypsin, NOT 0.05%) for 5 minutes. Piptette the cells/trypsin 5 times with a p1000 to break up any ES cell clumps. Transfer to a 15mL conical and spin cells (low speed, about 125g, 3min) through 8mL media (very important to get rid of trypsin as it makes the cells sticky for injection and compromises viability of the blastocysts). Resuspend in 2mL media, remove the gelatin from the new 6-well and place the cells in the gelatinized well for 30-35 minutes. The MEFS will sit down during this time, but ES cells will still be floating.
Transfer the floating ES cells (still have ~2mL) to a 15mL conical, resuspend up and down well (at least 10 times) using a cotton-plugged Pasteur pipette (preferred) or a p1000. Now count how many cells you have by taking a 10uL sample into the hemacytometer. Count all cells in a big box (there are 9 big boxes like a tic-tac-toe that contain other small boxes). You may need to count several big boxes to get an average if the # of cells in each box is below 30.
Calculate the number of cells in your 2mL sample:
ml in sample x average # cells in a box = total x 104 cells
average # cells in a box = cells/ml x 104 cells
|mL||Cells in a box||Total cells||cells/mL|
Use this information to dilute, concentrate, or freeze back some of your cells so that you have the optimal concentration of cells for your injectionist. Here is the best prep for them:
Label each of the cryovials with
Place the tubes on ice. The cells are now ready, so call x3-0675, ask for your injectionist and s/he can meet you near the Bio café in building 68.
Other info you should have already given to the injection facility (if not, write this down for them now so there are no questions/delays later):