ESCellCultureandManipulation3

Care and Handling of Feeder Layer Cells

STO/SNL cells are a derivative of the standard STO cell line. They are transformed with a LIF producing plasmid (low-level). They grow quite well, and look slightly more vacoulated than regular STO cells. This line was made available to use from the Bradfield lab.

STO Cells

• High glucose DMEM (-pyruvate,-glutamine)

• Newborn calf serum (heat inactivated 56°C for 30 min)

• 1X l-glutamine (add fresh every 2 weeks)

• 1X penicillin/streptomycin

1. Thaw rapidly at 37°C, rinse outside of vial with 70% ethanol

2. Use a lml pipet to transfer cells to a 15ml centrifuge tube, add 5ml STO/SNL media

3. Spin down cells for 2 minutes at 1000 rpm

4. Aspirate off media and resuspend cell pellet in fresh media, plate

1. Grow cells in 10 cm tissue culture dishes

2. Change media every 2 days

3. Split cells 1:10 as soon as they become confluent (usually ~ 4 days)

1. Aspirate all media from plate and add ~5 ml. incomplete PBS. aspirate. Repeat 2X. Add 3ml. trypsin-EDTA.

2. Put plate in 37°C incubator for 8-10 minutes

3. Tap plate to check that cells are loosened

4. Add T-E solution to an equal volume of media in a centrifuge tube.

5. Pipette back and forth a few times to insure a single-cell suspension. Spin 2 min at 2000 rpm.

6. Aspirate media, resuspend cell pellet in fresh media and plate. Usually cells are plated 1:8.

STO/SNL Cells

• High glucose DMEM (-pyruvate,-glutamine)

• Newborn calf serum (heat inactivated 56°C for 30 min)

• 1X l-glutamine (add fresh every 2 weeks)

• 1X penicillin/streptomycin

1. Thaw rapidly at 37°C, rinse outside of vial with 70% ethanol

2. Use a lml pipet to transfer cells to a 15ml centrifuge tube, add 5ml STO/SNL media

3. Spin down cells for 2 minutes at 1000 rpm

4. Aspirate off media and resuspend cell pellet in fresh media, plate

1. Grow cells in 10 cm tissue culture dishes

2. Change media every 2 days

3. Split cells 1:10 as soon as they become confluent (usually ~ 4 days)

1. Aspirate all media from plate and add ~2ml 1X trypsin, swirl so all cells are covered

2. Put plate in 37°C incubator for 5 minutes

3. Tap plate to check that cells are loosened

4. Add 3ml media to plate, pipet up and down several times to achieve a single-cell suspension

5. Transfer cells to a 15ml centrifuge tube, spin 2 min at 1000 rpm

6. Aspirate media, resuspend cell pellet in fresh media and plate

Trypsinize either regular or MitoC-treated STO/SNL cells as aboveAfter centrifuging resuspend cells in freezing media (STO/SNL media with 20% NCS and 10% DMSO)Volume should be approximately lml per plate of cells, aliquot to cryovials adding lml per vialFreeze vials immediately at -20°C for ~2 hr, then at -80°C overnight, transfer to liquid N2 the next morning.

Growth-Arrest for Feeder Layers

Split cells as described above. However, instead of plating, transfer the cells (in media) to a sterile centrfuge tube. Parafilm the tube, and place the cells in a small ice-bucket. Bring the cells down to the gammacell (you MUST be certified to use the gammacell. See Rex for information about certification). Place the cells in the sample cavity (don’t forget to sign in) and irradiate for 40 minutes. Wipe off the tube with 70% ethanol, and place the tube back in the hood. Plate the cells, using 1 full plate of cells for 1 plate of feeders. One 10cm2 plate makes enogh feeder cells for another 10cm2 plate, 2 24-well plates, or 2 96-well plates. Check the cells and change media (to ES cell media) the next day. It is not unusual to see some death, but normally the cells spread out enough that this does not cause problems. You want the cells to cover the entire plate, confluency is preffered. You do not wish plated ES cells to be exposed to open surfaces on the plate (although they generally only stick to the feeder cells anyway).