HEK 293 EXPRESSION SYSTEM

Storage of Transfected HEK 293

When you have stable cell line, which expreses the protein of your interest the cells should be stored by freezing at -80°C( up to 3 months) or in liquid nitrogen(~-196°C) for several years.

Reagents:

Media:

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)

DME-HG/ FBS/ Pn/ Str/ containing 10% DMSO (kryoprotector)

Trypsin (0.05% solution with 0.5 mM EDTA)

Phosphate Saline Buffer (PBS), pH 7.0-7.2

*The amount of time the cells spend in trypsin should be as short as possible. Keep that in mind when going through this procedure.

Procedures:

• Aspirate the media

• Wash the plates with ~10 ml PBS

• Trypsinize the cells with 3 ml trypsin

• Put the trypsinized cells in15 ml Falcon tubes with 7-10 ml DME-HG/ FBS/ Pn/ Str( the trypsin gets partly inhibited)

• Centrifuge them for 10 min at 600 RPM

• Aspirate as much of the supernatant as possible

• Resuspend the cell pellet gently, but thoroughly, in 3 ml DME-HG/ FBS/ Pn/ Str/ 10% DMSO

• Aliquot the suspension in freezing vials placed in a styrofoam box (app. 1 ml in each vial) and place immediately in the freezer (-80°C)

• After 24h the vials should be placed in liquid nitrogen.