Reagents:

  • Appropriate culture media (i.e. cRPMI for T cell lines, cDMEM for attaching cells) warmed in 37° C waterbath.

  • cRPMI = RPMI + 10% FBS+ L-Glut + P/S.

  • cDMEM = DMEM + 10%FBS+ L-Glut + P/S + HEPES

  • Sterile PBS warmed in 37° C waterbath.

  • Antibiotics/proteins/growth factors as needed

 

Protocol:

 

Check all cell lines under the microscope.

For Attaching Cells

  1. Draw off all supernatant with a pipette. Take care not to disturb the cell layer.

  2. Wash once, gently, with PBS to remove any debris.

  3. Add just enough trypsin to cover the cell layer (~2.5mL in T75, ~0.5mL in T25).

  4. Incubate for 2 minutes MAX at RT.  Shake and tap occasionally to verify that the cells are releasing.

  5. Add 10mL of cDMEM and mix the cells thoroughly by pipetting up and down until there are few clumps.

  6. Draw all of the cell suspension into the pipette, and then leave 1mL in the flask.

  7. Discard the rest or save for an experiment.

  8. Add 25mL of fresh media.

  9. Write new passage number on flask. Take care not to leave the flask upright, as the cells will begin to reattach with the new media.

 

For Suspension Cells

  1. With a pipette, mix the cells thoroughly by pipetting up and down, making sure to rinse the side of the flask.

  2. Draw all of the cell suspension into the pipette, and then leave 1mL in the flask.  Discard the rest or save for an experiment.

  3. Add 10mL of fresh media.

  4. Write new passage number on flask.


Appropriate culture media

 

GHOST cells

  • DMEM + 10% FBS + L-Glut + P/S + HEPES

  • 500 mg/ml G418

  • 100 mg/ml Hygromycin B (reduce to 50mg/ml if cells appear too sensitive)

  • Coreceptor expressing cells only (i.e. not parental): 1mg/ml Puromycin

 

U87 cells

  • DMEM + 10% FBS + L-Glut + P/S + HEPES

  • 300 mg/ml G418

  • Coreceptor expressing cells only (i.e. not parental): 1mg/ml Puromycin

 

HELA cells

  • DMEM + 10% FBS + L-Glut + P/S + HEPES

  • Coreceptor expressing cells only (i.e. not parental): 0.4mg/ml Puromycin

 

For specifics on appropriate handling and waste procedures please see the online chemical SOPs or our waste and spill notebook located in room 352.

Department of Microbiology
School of Medicine
University of Washington
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