293 EXPRESSION SYSTEM
Cultivation of Human Embryonic Kidney 293 Cells
293 cells are stored for long periods of time in liquid nitrogen (-196oC) frozen in medium (e.g. DMEM), containing 10% DMSO.
Medium:
500 ml Dulbecco's Modified Eagle (DME-HG)
50 ml Fetal Bovine Serum (FBS) - 10 % of the medium volume
5 ml penicillin/streptomycin (10 000U/ml Pn and 10 mg/ml Str)-1x final concentration
Trypsin (0.05% solution with 0.5 mM EDTA)
Phosphate Saline Buffer (PBS), pH 7.0-7.2
Procedures:
Starting from a Cryotube:
Prewarm the medium at 37°C and fill one 15 cm dish with 25 ml DO NOT USE MEDIA THAT CONTAINS HYGRO. , G418, etc. AT THIS POINT
Rapidly thaw the cells in your hand, gently pippet up and down to thaw any remaining ice before distributing in a dish filled with warm media. Rock the dish to distribute the cells. Do not swirl-if you do they will all clump towards the center of the plate.
Change the medium after 24 hrs or once the cells have attached.
Splitting Cells (all solutions have to be prewarmed):
Prepare a new dish with fresh medium. (25 ml for 15 cm plates, 10 ml for 10 cm plates)
Aspirate the old medium from the dish.
Wash the cells carefully with PBS to remove residual medium and FBS which can inhibit the trypsin.
Trypsinize cells by adding 3-5ml of trypsin to the dish and incubate at 37°C (or RT, it depends on its activity) until cells have detached (usually 2-5 minutes).
Take a fraction of the cell solution and transfer it to the new dish (BSA in medium inhibits the trypsin).When splitting confluent 293 cells in a 1:10 ratio, confluence is reached again after 3-5 days.