Determining the Role of Neutrophil-Like Cells in Lymphoma Cell Sensitivity to a Test Drug

To set up a 3D co-culture, add 50,000 RL lymphoma B cells alone, or mix with HL60-differentiated cells in sterile 15-milliliter tubes.

Spin down the samples and resuspend the pellets in 300 microliters of basement membrane matrix using a 1-milliliter pipette tip with the opening cut to 2 to 3 millimeters. Next, incubate 300 microliters of the cells in each well of a 24-well plate for 30 minutes at 37 degrees Celsius and 5% CO2.

At the end of the incubation, add 1 milliliter of complete RPMI medium to each well, and return to 3D cultures to the incubator for another seven days with medium changes every two days. On day 5, add 10 nanomolar vincristine to the cultures.

After one week of culture, aspirate the medium and wash each well 2 times with 1 milliliter of ice-cold PBS. Then, add 3 milliliters of ice-cold PBS with EDTA to each well, and scrape the bottom of the wells with a 200-microliter pipette tip to detach the gels. Shake the plate gently on ice for 30 minutes. Then, transfer the cell suspensions to individual sterile 15-milliliter tubes for gentle shaking on ice for another 30 minutes.

Confirm the appearance of a homogeneous cell suspension. Then, spin down the cells, followed by a PBS wash, and resuspend the pellets in fresh PBS with FBS. Finally, label the cells with the appropriate antibodies and viability dyes as just demonstrated, and analyze the RL and HL60-differentiated cells by flow cytometry using the illustrated gating strategy.