- 传世开服网
ES Cell Culture and Manipulation
-
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 l 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 (~51) 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 200l trypsin for ~10 minutes. Add 800l 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 (~140l to feeder well and 601 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 (7001 to feeders and 300l 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 4001 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
- 共2页:
- 上一页
- 1
- 2
- 下一页