Galli Lab– In the Department of Pathology

WEHI-3 cell culture and generation of conditioned medium for BMCMC culture (PDF)

Materials Needed

WEHI-3 cells (myelomonocytic leukemia, macrophage-like, Balb/C Mouse cells,
    available from ATCC: TIB 68)
T-175 flasks, other flasks as needed
CellTiter 96® AQueous One Solution Cell Proliferation Assay (Promega Cat.#
    G3582): a colorimetric assay for cell viability (optional)

Medium

       CMLESS Medium* (basic complete medium for growth factor-independent cells)
       For 500 ml: 10% Fetal Bovine Serum, heat inactivated
2 mM L-glutamine (add fresh as needed)
5 x 10-5 M ß-mercaptoethanol
1% antibiotic/antimycotic
bring up volume to 500 ml with DMEM
*Filter all through a 0.2 µm filter to sterilize
*Filter all through a 0.2 µm filter to sterilize

Cell maintenance

  1. Maintain a stock of WEHI-3 cells at a concentration of 105 cells/ml in CMLESS medium, 37°C, 5% CO2.
  2. Check cell counts and feed cells twice weekly. For routine maintenance, spin down cells at 1000 RPM for 10 minutes, aspirate supernatant and resuspend in fresh medium at 105 cells/ml.

Production of conditioned medium

  1. Set up T-175 flasks (as many as needed) of WEHI-3 cells at 105 cells/ml.
  2. Incubate for 2-4 days without feeding until the cell concentration is 6 x105 to 1 x106 cells/ml. If the cells have not reached the target concentration by day 4, use a different flask.
  3. Spin down cells at 1000 RPM for 10 minutes. Transfer the supernatant to a new tube and discard the cells.
  4. Spin down supernatant again at 2500 RPM for 15 minutes.
  5. Filter supernatant through at 0.45 µm filter into a sterile bottle.
  6. Remove approximately 5 ml of each batch for testing (if desired), and store remainder in 200 ml aliquots at -20°C for later use.

WEHI-3 conditioned medium testing (optional)

  1. For each batch of WEHI-3 conditioned medium (WEHI-3 CM) produced, prepare samples for testing by diluting 1 ml of conditioned medium in 1.5 ml CMLESS medium (40% WEHI-3 CM solution).
  2. Prepare cells from the CellTiter 96® AQueous One Solution Cell Proliferation Assay by taking 2.5 x106 cells in a 15 ml centrifuge tube, spun down at 800 RPM for 8 minutes at room temperature. Resuspend cells in 5 ml CMLESS medium so that final cell concentration is 5 x105 cells/ml.
  3. For each batch tested, add 50 µl of 40% WEHI-3 CM into a well of a 96 well flat-bottomed plate. Place plate in incubator at 37°C, 5% CO2 to equilibrate for 1 hour.
  4. Add 50 µl of CellTiter/CMLESS cell suspension from step 2 to each well and shake gently to mix. The final cell concentration should be 2.5 x104 cells/well.
  5. Incubate the plate at 37°C, 5% CO2 for 72 hours.
  6. After incubation, add 20 µl of AQueous One Solution reagent into each well then incubate for 2-4 hours.
  7. Record absorbance at 490 nm in a 96 well plate reader.

 

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