NIH/3T3 Cell Culture Media Preparation

From LPDwiki

About this protocol: This protocol outlines the steps and materials involved in creating sterile culture medium used in maintaining cultures of NIH/3T3 murine fibroblasts.

Necessary Materials

1. Dulbuecco's Modified Eagle Medium (DMEM) with L-glutamine and sodium pyruvate:

  • DMEM is purchased from Invitrogen (11995-065) and should be stored in 4°C upon arrival.

2. Heat-inactivated Fetal Bovine Serum (FBS)

  • FBS is purchased from Invitrogen (10082-139).
  • Upon arrival FBS should be aliquoted to 50 mL and stored at -20°C

3. Penicillin-Streptomycin (Pen/Strep)

  • Pen/Strep is purchased from Invitrogen (15140-122).
  • Upon arrival Pen/Strep should be aliquoted to 5 mL and stored at -20°C

4. Autoclaved Glass Bottles (2x)

  • Autoclaving is done by UC Davis Veterinary Medicine Central Services located in Haring Hall.

5. 0.2 um Vacuum Bottle Top Filter (1x)

  • Bottle top filters can be purchased from UC Davis Veterinary Medicine Central Services located in Haring Hall or Sigma Aldrich (CLS430049).

Procedure

Important: The Biosafety Cabinet (BSC) must be used for the preparation of NIH/3T3 cell culture media. Please follow the Biosafety Cabinet Start-up procedure before beginning this protocol.

1. Thaw one aliquot of both FBS and Pen/Strep overnight in 4°C before starting this protocol.

Inside the BSC

2. Place both autoclaved bottles and sterile filtering unit into the BSC.

3. Add 445 mL of DMEM solution into one autoclaved bottle (Bottle A).

4. Add 50 mL of FBS to the 445 mL of DMEM solution (Bottle A).

5. Add 5 mL of Pen/Strep to the bottle containing both FBS and DMEM (Bottle A).

6. Agitate the 500 mL of culture media to mix (via pipetting).

7. Place the vacuum filter on the second autoclaved bottle (Bottle B) and turn the vacuum on.

8. Slowly pour the media from Bottle A through the filter into Bottle B.

9. Turn the vacuum off and detach the vacuum filter before discarding in the biological waste bin.

10. Cap both Bottle A and B tightly.

Outside the BSC

11. Label Bottle B with: (write on tape not on the glass)

  • Date made:
  • Solution Name: NIH/3T3 Cell Culture Media (DMEM, 10% FBS, 1% Pen/Strep)
  • Your name:

12. Place the cell culture media into 4°C storage.

13. Place the used bottle (A) into the sink for cleaning.

Cleaning Up

1. Ensure the remaining DMEM is tightly capped, marked sterile, and place into 4°C for storage.

2. Dispose of used conical tubes and pipettes into the biological waste bin.

3. Thoroughly rinse out the used bottle and hang up to dry.

4. If you are done with work in the BSC, follow the Biosafety Cabinet Shutdown procedure.