Splitting 293T cells

8/19/04

Medium:

500mL DMEM (high glucose)

50mL FBS

5mL pen/strep

5mL glutamine

You need to split the cells before they are overgrown. A 1:10 split will need to be resplit in about 3 days, 1:20 in about 4 days and 1:40 in about 5 days.

Cells need to grow at 10% CO2, so use lower incubator on the right.

Prewarm medium, 1X Ca-Mg free HBSS, and trypsin (can use the trypsin in the 4°) for at least 20’ in tc water bath.

Remove medium from cells and wash 2X with 5mL Ca-Mg free HBSS. Make sure to spray the solution on the side on the plate not right down on the cells so they don’t wash off. Minimize the time the cells are dry after removing the medium.

Remove HBSS and add 2mL trypsin. Return plate to incubator for 3-5’.

Gently shake plate to see that cells are coming up. You can also watch cells come off the substrate by looking on the microscope.

Leave trypsin on and add 10mL serum-containing medium. Spray around the plate to spray all the cells off the plate. Move to 15mL tube and spin cells down 5’ at 2K in tc centrifuge.

Aspirate medium and add 10mL fresh serum-containing medium. Resuspend cells 1X with 10mL pipette, then 1X with a P200 tip on the end of the 10mL pipette.

If you are plating for virus, use hemacytometer to count cells. All the cells in 25 large squares x 10 to the 4^th equals the number of cells you have per mL. For virus, plate at 0.5x10 to the 6^th cells per mL.

For expanding the cells, plate as many as you need at 1:10 (resuspend cells in 10mL, then put 1mL of that into 10mL fresh medium on a new plate)

. These will be confluent and ready to split again in 3 days.

For carrying, plate one at 1:20 and one at 1:40 (so if one plate resuspended in 10mL medium, use 0.5 or 0.25mL of that solution on a new plate in 10mL). These will need to be split again in 4-5 days.

For transfection, plate as many as you need at 1:3 or 1:5 (so if one plate resuspended in 10mL medium, use 3.3mL or 2mL in 10mL new medium on a new plate.) These numbers are approximate, so don’t worry if it is not exact. These cells should be 50-70% confluent the next day at the time of transfection.