Picking ES cell clones
One or two days before picking colonies prepare 24-well plates of feeders. You can also use alternate protocols that utilize 96-well plates, small tissue culture dishes, or 6 well plates, if you prefer. To pick clones you will need: fresh trypsin-EDTA, a P-200 pipetman and sterile yellow tips (preferably barrier, or plugged tips), a sterile 96well round-bottom plate, plenty of ES cell media, and an inverted microscope in a hood. Aspirate the REF media from the 24-wells and add 2ml of ES cell media to each well. Add 40 µl trypsin-EDTA to as many wells of the 96-well plate as you plan to pick clones. Working with one 10cm plate at a time, wash the plate with 2ml trypsin and then add another 2ml. Using the P-200 pick up a small amount trypsin (~5µ1) from the edge of the plate then pick up a well isolated colony in a minimal amount of trypsin. You can dislodge the colony slightly with the pipette tip. Transfer the colony to a well of the 96-well plate and number this well so you don't mix clones together. Repeat for each colony to be picked from this plate. Pick all sizes of colonies but you will have to work fast before the plate dries out. If clones become too loose and start to move around you can reduce trypsin to lml or skip the trypsin rinse. After picking the first 1/3 of the clones they must be broken up into a single-cell suspension -- this is very important because any clumps of cells will begin to differentiate, often within the first few days! Use the P-200 with yellow tips to pipette each clone many times (10-20X, try to keep number of bubbles down) and check individually under the 4X microscope to be sure no clumps are left. Transfer each disaggregated clone to a numbered 24-well feeder (the 2ml media will neutralize the trypsin). Notes: 1.) Change yellow tips for each clone at each step, you don't want to cross contaminate them 2.) The ES cells can take more pipetting and longer times in trypsin than you would expect, however work quickly and with one plate at a time so trypsin exposure doesn't become excessive 3.) I usually check all clones visually under the scope ~ every 2 days and if some are growing clumpy they may need to be split 1:1 before they are actually confluent.
Expansion of ES cell clones
Change the ES cell media of each picked clone the following day (now you only add lml per well) and every 1-2 days until the clones begin to become confluent (as evidenced by yellowing media) in ~5 days. When a clone is confluent it is trypsinized and split to one well of a fresh 24-well feeder and a gelatinized well of a 6-well plate, be sure to make the 24-well feeders ahead of time. Trypsinize by rinsing with 0.5ml trypsin-EDTA, aspirate and add another 200µl trypsin for ~10 minutes. Add 800µl ES media with a lml pipette with a yellow tip and pipette several times to break up cells. Transfer to a sterile l.5ml eppendorf tube and centrifuge ~2min at 3000rpm. Aspirate media and use a P-200 to resuspend pellet in 200,u1 ES media, transfer to feeder and gelatin wells (~140µl to feeder well and 60µ1 to gelatin). Feed the 24-well feeder plate the following day and by the next day the cells should be yellowing the media and are ready to be frozen (see freezing protocol below). I don't usually refeed the gelatin plate, just scrape the cells for DNA in several days when media is yellow. Note: If you have picked many clones the above method may be impossible to follow time-wise. A way to shorten the protocol is to eliminate the centrifuge step and just add the lml trypsin/ES mix to the two plates (700µ1 to feeders and 300µl to gelatin). Even this small amount of trypsin may make the clones grow a bit slower initially but if you change the media early the next day they seem to do OK and this saves a lot of time.
Freezing of ES cell clones in 24-well plates
When the 24-well feeder plates of clones begin yellowing the media they are ready to be frozen (~2 days). Make ES cell freezing media ahead of time and chill in fridge or on ice Also prechill a small styrofoam box at -80°C (the boxes NEB ships enzymes in are a perfect size, or you can hollow out the styrofoam trays from 15ml centrifuge tubes and make a sort of box out of them. This box is identical to the ES cell freezing box described above). Aspirate the media from a 24-well plate of clones and add 400µ1 prechilled freezing media to each well Wrap the plate well in double parafilm; if freezing multiple plates place on ice until all are done. As quickly as possible move plates into prechilled box in -80° freezer. Freeze plates several hours or overnight at -80° then move box to a -135°C freezer for long-term storage (i.e. liquid nitrogen, if possible). Cells can be kept long-term at -80° but it should be in a freezer that is rarely opened since the worst possible thing is repeated freeze-thaw!
Scraping ES cells from gelatin for DNA
When cells are confluent and media is good and yellow scrape cells to make DNA. Aspirate media and carefully rinse each well 2 times with lml of incomplete PBS. Add another lml PBS and scrape cells with a rubber policeman, transfer to an eppendorf tube and freeze at -20°C until ready to extract DNA.
Gelatin treatment of Tissue-culture plates
Make a 0.1% solution of gelatin (Sigma # G-1890) in Milli-Q water and autoclave, cool. Add enough gelatin to each plate, or well of a plate, to cover the bottom and let sit at least 5 minAspirate and add STO/SNL media.
Extraction of DNA from ES cells
Rapid Preparation of DNA from ES cells in 24-well tissue culture dishes
This simple method based on a protocol described by Miller et al. (1988) involves salting out cellular proteins with a saturated NaCl solution. It does not require extraction with phenol. Sufficient DNA can be obtained by this method for screening by Southern blot analysis (3-5 µg)
Materials And Equipment
Prepared 24-well tissue culture dishes containing nearly confluent ES
cell cultures
Phosphate-buffered saline (PBS) (incomplete)
Lysis buffer (150 mM NaCl, 2 mM EDTA, 1% SDS, 20 mM Tris-HCl [pH 8.0],
µg/ml proteinase K)
microfuge tubes
Solution of saturated NaCl
and 100% ethanol at room temperature
automatic micropipettor with a disposable tip or glass rod
ml of TE buffer
Caution: Wear a mask while weighing SDS.
Procedure
1. Aspirate medium from each well containing nearly confluent ES cells and rinse the cells once in PBS. Resuspend the cells in 200 yl of Iysis buffer. Incubate the dish for several hours to overnight at 55°C.
2. Transfer to 1.5-ml eppendorf tubes. After digestion with lysis buffer is complete, add 100 µl of saturated NaCl to each tube and shake vigorously (do not vortex, to avoid shearing fragile genomic DNA).
3. Centrifuge the tubes at 3000g in a microfuge for 15 minutes. Transfer the supernatant containing DNA to a fresh 1.5-ml polypropylene tube and add 2 volumes of ethanol at room temperature. Invert the tube several times until the DNA precipitates and then remove it with the disposable tip of a 200-µl pipetteman or with a glass rod. Rinse the pellet in 70% ethanol and resuspend it in 50 µl of TE. Allow the pellet to dissolve at room temperature for several hours.
Preparation of LIF Conditioned Media
The 720 LIFD cell line has been transformed with a plasmid that causes them to secrete a high level of LIF. For unknown reasons, this causes the cells to grow very slowly. They are also more easily contaminated (in my hands) than normal STO cells. Also, the cells will look sick, compared to normal STO cells. They tend to be slightly smaller, and much more vacoulated.
Media
GIBCO a-MEM (-l-glutamine)(720LIFDs are fussy about the brand of media, they don't like Sigma)
10% Heat-inactivated calf serum
1X Penicillin/streptomycin
DO NOT add l-glutamine, the cells don't like it!
Growing 720LIFD cells
Grow cells on gelatinized 10cm tissue culture plates (see STO/SNL protocol for gelatinizing). Change media every 2 days, the cells will grow slowly, and look messed up. This is normal for this cell line. Split cells as soon as they become confluent.
Harvesting LIF conditioned media
When cells are almost confluent (~80-90%) change the media. The following day collect the conditioned media in a 50ml centrifuge tube(s)Spin at 2000 rpm for 5 min to remove any cells. Filter through a 0.2µ filter and aliquot into cryovials or parafilmed-centrifuge tubes. Store the stock LIF at -80°C. You can usually add fresh media to these cells and harvest a second batch of LIF the next day. Throw these cells out when you are done as, they have now been confluent for ~2 days. LIF conditioned media CAN be stored short term (a few moths) at -20°C.
Titering LIF
Plate 1 x 104 feeder-free ES cells in each of the wells of a gelatinized 6-well in ES media without LIF. At plating add LIF at 1:100, 1:500, 1:1000, 1:2500, 1:5000 and 1:10,000 dilutions from stock. Check for differentiation over a 1 week period. Select the lowest dilution than that which allows any amount of visible differentiation. Usually 1:2500 or 1:5000 is best(If the ES cells are becoming confluent in less than 1 week you may need to plate a smaller number)
Notes: The cells can occasionally be reselected in 0.1mM methotrexate to make sure they are still stably transformed for the LIF transgene. When growing in methotrexate the calf serum must be dialyzed.
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