Inventory of Cell Culture Materials for HEK 293 and BHK Cells

1.D-MEM/F12 (with 1X with L-GLU; Sodium Bicarb.; Pyridoxine HCl)

Manufacturer:INVITROGEN

Cat No:11320-033

CellCenter:Special Order ($16)

2.Fetal Bovine Serum (Qualified; Orig USA)

Manufacturer:INVITROGEN

Cat No:26140-079

CellCenter:7127 may work or Special Order ($100)

3.Penicillin-Streptomycin (100X)

Manufacturer:INVITROGEN

Cat No:15140-122

CellCenter:1188

4.L-Glutamine (200 mM, 100X)

Manufacturer:INVITROGEN

Cat No:25030-081

CellCenter:1187

5.Trypsin/EDTA (0.25%, 1 mM EDTA)

Manufacturer:INVITROGEN

Cat No:25200-056

CellCenter:1236

6.Vitamin K1 (10 mg/mL; Phytonadione Injection, USP)

Manufacturer:Abbott Laboratories

Cat No:NDC 0074-9158-01

**Order from CHOP pharmacy**

7.Dulbecco’s Phosphate Buffered Saline (PBS; 1X; without CaCl2, MgCl2)

Manufacturer:INVITROGEN

Cat No:14190-136

CellCenter:1115

8.Genticin (G418 Sulfate)

Manufacturer:INVITROGEN

Cat No:11811-031

CellCenter:1124

9.Insulin-Transferrin-Sodium Selenite Supplement (ITS, 50 mg)

Manufacturer:Roche

Cat No:1074547

CellCenter:3196

10.Freezing Mix (95% Calf Serum/5% DMSO)

CellCenter:1122

  1. Opti-MEM-reduced serum hybrid medium (500 mL)

Manufacturer:INVITROGEN

Cat No:31985-070

CellCenter:1163

12.Albumax I (25 g)

Manufacturer:INVITROGEN

Cat No:11020-021

CellCenter:Special Order

13.Penicillin-Streptomycin-Fung (100X)

Manufacturer:INVITROGEN

Cat No:15240-062

CellCenter:1167

  1. Triple Flasks (filter)

Manufacturer:NUNC/FISHER

Cat. No.12-565-25 ($308 case of 32)

Useful web sites:

Media for HEK 293, BHK, and COS-1 Cells

Untransfected Cells without G418 Selection: (“MEDIA”)

  • 500 mL of D-MEM/F12
  • 50 or 25 mL of FBS (10%, final; can use 5% as well)
  • 5 mL of L-Glutamine
  • 5 mL of Penn/Strep/Fung
  • 300 l of Vitamin K (6 g/mL, final) [ADD ONLY IF PRODUCING VITAMIN K-DEPENDENT PROTEINS]

**Store at 4C; Good for at least a few weeks.

Transfected Cells with G418 Selection (Cells containing NEO Vector, stable cell lines; G418 Media): (“SELECTION MEDIA”)

  • 500 mL of D-MEM/F12
  • 50 mL of FBS (10%, final, can use 5% as well)
  • 5 mL of L-Glutamine
  • 5 mL of Penn/Strep/Fung
  • 300 l of Vitamin K (6 g/mL, final)[ADD ONLY IF PRODUCING VITAMIN K-DEPENDENT PROTEINS]
  • 0.32 g of G418 to 500 mL of Media (450 g/mL, final; may use higher amounts in certain situations, adjust accordingly)
  • **Note: The active concentration of the G418 Stock is ~708 g/mg of solid, depending on lot. Need to look at bottle.

Note on preparation of G418 Selective media: Weigh out desired amount of G418 and place in 50 mL conical. Take 10 mL of D-MEM/F12 and dissolve G418; Note color change to yellow (acidic). Adjust pH with a few drops of 6M NaOH. Sterile filter solution (0.2 m) into 500 mL bottle of media.

Media for Large Scale Production of Recombinant Factor X:

  • 500 mL of D-MEM/F12
  • 0.5 mL of ITS (1X)
  • 5 mL of L-Glutamine
  • 5 mL of Penn/Strep
  • 300 l of Vitamin K (6 g/mL, final)

Note on preparation of ITS: Dissolve ITS (50 mg) in 5 mL of sterile water (1000X stock). Sterile filter (0.2 m). Store in 0.5 mL aliquots at -20C. Good for several months.

Media for Large Scale Production of Recombinant Factor V/V-DT: (“EXPRESSION MEDIA”):

  • 500 mL of DMEM/F12
  • 0.5 mL of ITS (1X)
  • 5 mL of L-Glutamine
  • 5 mL of Penn/Strep/Fung
  • 5 mL of Albumax (1.5 mg/mL, final)
  • 1 mL of 1M CaCl2 (0.2 µm filter; 2 mM, final)

Note on preparation of ITS: Dissolve ITS (50 mg) in 5 mL of sterile water (1000X stock). Sterile filter (0.2 m). Store in 0.5 mL aliquots at -20C. Good for several months.

Note on preparation of Albumax: Dissolve Albumax (25 g) in 166 mL (150 mg/mL) of MilliQ water. Mix slowly for 30-40 min @ RT. Filter sterilize (filter may need to be changed 2-3X). Store at -20 C in 5 mL aliquots.

Maintaining HEK 293, BHK, and COS-1 Cells

** We maintain cells in T25 Flasks, which contain 10 mL of media.

To split cells: (All solutions should be warmed to 37C)

  • aspirate media
  • wash cells with 10 mL of PBS
  • aspirate PBS
  • add 0.5-1 mL of trypsin/EDTA to flask
  • mix and let sit 2-5 min (until cells lift)
  • add 9 mL of fresh media (the serum neutralizes the trypsin)
  • take 1 mL (or less) of this cell solution and place in fresh flask with 8-9 mL of media
  • place cells back in 37C incubator