Lab 8: Passaging Cells

Objective:
In biology, a subculture is a new cell or microbiological culture made by transferring some or all cells from a
previous culture to fresh growth medium. This action is called subculturing or passaging the cells. Subculture is
used to prolong the life and/or expand the number of cells or microorganisms in the culture.

Supplies:
 Flask containing cell culture  1x T-____ Cell Culture Flask
 1x sterile ___mL centrifuge tube  50mL of sterile DMEM
 10 mL sterile trypsin  50 mL sterile PBS

Prep:
1. Put on PPE, prepare the BSC
2. Label the T-___ flask with: the cell type, workspace #, students initials XXX, the date, passage #
3. Label the centrifuge tube with workspace #
4. Spray supplies with 70% ethanol and quickly move into BSC

Waste Management:
1. Keep serological pipette packaging separate from biohazard waste. Dispose in standard trash

Other BSC working tips:

1. Keep clean and dirty items separate
2. Keep containers covered / closed as much as possible
3. Work in the center of the BSC, DO NOT place sterile items near the front grate

Protocol:
1. Warm reagents in the water bath (media, PBS, trypsin). Record source of each reagent before moving
into BSC
2. Retrieve your team flask from the incubator and use microscopes to visually examine the level of
confluence in your flask. Note the confluence in your notebook. Are there any other observations
(irregularities?)
3. Move cell culture flask into BSC
4. Aspirate media
5. Using a _____________ add _____ mL of PBS to the flask
6. Gently rock the PBS over the cells to wash them
7. Aspirate PBS
8. Using a _____________ add ______ mL of trypsin
a. This volume should be enough to cover bottom of flask
9. Gently rock the trypsin over the cells
10. Place the flask in the incubator for 5 minutes
a. This time amount may be different depending on cell type!
b. If you trypsinize too long, you may kill the cells!
11. Use the microscope to check that cells are detached

BME 3323L Cellular Engineering Laboratory

Rowlinson, Spring 2018
 a. Detached cells should look round and floating
12. Quickly add 2 X (volume trypsin) mL of media to inactivate the
trypsin Helpful Hints!
a. For example add 12 mL of media to 6 mL trypsin to
 Don’t over-fill pipettes when
properly inactivate the trypsin
using the Pipette-Aid
13. Gently rock the flask, followed by pipetting up and down,
 When resuspending, pipette
flushing the entire bottom of the flask
slowly back and forth to
14. Using the same serological pipette, transfer all contents to a ___
break up the cell pellet and
conical tube
try to avoid bubbles!
15. Balance and centrifuge samples at 1000 RPM for 5 minutes
a. Wait for other groups to add samples
b. Water weights located below centrifuge
16. Confirm cell pellet is present
17. Aspirate trypsin/media supernatant
18. Resuspend the cells (the pellet) in 5mL of fresh media
a. Either pipette up and down or vortex
b. No visible cell clumps should be seen
19. Move ____ % of cell suspension to new T-____ flask
20. Add additional fresh media so that the total volume in the T-____ flask is
correct
21. Using microscope, confirm cells present in new flask
22. Place cell culture in incubator

Clean-Up:
1. Put pipettes and accessories back on shared table
2. Clean BSC
3. Clean vacuum system line with 70% EtOH (reuse conical tube and place back next to stock jug)
4. Dispose of waste properly
5. Bring paper protocols with you

** In your lab notebook, please record which team member did which step of this protocol

BME 3323L Cellular Engineering Laboratory

Rowlinson, Spring 2018