Reagents:

DME-HG/ FBS/ Pn/ Str, which contains:

500 ml Dulbecco's Modified Eagle (DME-HG)

50 ml Fetal Bovine Serum (FBS) - 10 %

5 ml penicillin/streptomycin (10 kU/ml Pn and 10 mg/ml Str)

Trypsin (0.05% solution with 0.5 mM EDTA)

Phosphate Saline Buffer (PBS), pH 7.0-7.2

***The roller bottles used are ribbed, with ~ 1700 cm²of surface area-If you want to use bottles with 2200 or more cm² surface area-you must increase the amount of cells you innoculate each bottle with. This is very important if you wish to avoid cell patches that will degrade the quality of your culture.

Procedures:

Aspirate the media (it might be harvested also in order to collect your protein)

Pour directly in each roller bottle 350 ml of prewarmed media. Place the roller bottles in the incubator and let them roll for several minutes so if any damages or leaks are present they might be noticed and bottles can be discarded.
Wash each plate with appr.10 ml PBS
Trypsinize the cells with 5 ml trypsin ( do not trypsinize more than 5 plates at a time)
Pipette the suspension from each plate into one roller bottle
Put the roller bottles in the incubator and set the rpm at 0.3 - 0.5 rpm
When the cells are attached onto the bottles' surface (app. after 24h), increase the rotation to ~1rpm.
Change the media once the cells reach confluence. At this point we usually change into serum free media (if that's what is called for), or just add fresh media and continue the culture.

***If using serum free media-the first media should be discarded once the cells reach confluence and then each bottle should be washed free of the remaining media with 50ml of serum-free media. After washing, add back app. 250-300ml of media lacking serum. Harvest the serum free media after 2 days. Repeat if the cells appear healthy.