Culture Passaging

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About this protocol: This protocol outlines the steps and materials involved in passaging cultures of NIH/3T3 murine fibroblasts.

Necessary Materials

1. 1X Trypsin Solution

2. Phosphate Buffered Saline (PBS)

  • PBS[+] and PBS[-] are purchased from Invitrogen (GB122).
  • PBS should be stored at 4°C
  • Use only PBS solutions marked STERILE for this protocol

3. NIH/3T3 Cell Culture Media

4. Cell Culture Flasks


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

Preparing Culture Flasks

1. Turn on the 4°C cooled centrifuge.

Inside the BSC

2. Fill cell culture flasks with NIH/3T3 culture media

Media Amount per Flask Size
T75 12 mL
T25 4 mL

3. Ensure culture media is spread over the entire base of the flask.


Inside BSC

1. Aspirate the culture media from flask to be passaged.

2. Add 10 mL PBS[-] to the flask.

3. Wash cells by gently tilting the flask back and forth.

4. Aspirate the PBS[-] from the flask.

5. Add 2 mL of 1X Trypsin Solution to the flask.

6. Ensure trypsin covers the entire culture surface.

Outside BSC

7. Place flask into 37°C (5% CO2) incubator for 3-5 minutes.

8. Inspect flask under microscope to ensure cells have separated from the culture surface.

Inside BSC

9. Add 3 mL NIH/3T3 culture media to the flask (this halts the trypsin from further damaging the cells)

10. Transfer the 5 mL cell suspension to a 15 mL conical tube.

Outside BSC

11. Centrifuge the cell suspension for 3 minutes at 1200 rpm at 4°C

Inside BSC

12. Aspirate all liquid from the conical tube, leaving the cell pellet.

13. Re-suspend the cells using 2 mL of NIH/3T3 culture media. Pipette gently to break up pellet.

14. Calculate the appropriate dilution and cell suspension to each culture flask.

15. Label flask with:

  • Type of cell:
  • Passage number:
  • Date of passaging:
  • Your name:

16. Spread cells over the culture surface by rocking flask back and forth then side to side. (Take care not to get any media on the cap!)

Outside BSC

17. Place new flasks into the incubator.

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.