GROWTH MEDIUM (for both lines):

DME High Glucose (w/ L-Glutamine)
1.5 g/L sodium bicarbonate
10%FCS
replace or add medium 2-3X/week.

(Note: DME High Glucose is DMEM with 4.5 g/L glucose).

Split 1:3 (probably not enough) to 1:6, about every other day, or 2-3x /week.

Use Scraper, or else Suck off sup and add back 5ml of 1mM EDTA per 750 cm2 flask, let sit a couple minutes, pipet up and down,, spin, get rid of EDTA, and plate. (Make sure the cells came off-- you might have to scrape themoff anyway).

37°ree;C, 5%CO2 incubator .

Cells frozen down (6/2000) are in 1 part medium to 2-3 parts of freeze medium.

Freeze medium: 90% FCS , 10% DMSO. Number of cells per vial: ~1/3 of a confluent 750 cm2 flask.

Don't add antibiotics to the growth medium. The cells grow fine without it, and if there is an infection you want to know about it so you can throw away the flask rather than risk the possibility of keeping the bacteria around but suppressed to low levels by antibiotic.

Shirley Reynolds