ESCellCultureandManipulation2

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.

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

Procedure

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.

Growing 720LIFD cells

Harvesting LIF conditioned media

Titering LIF