This study investigates the effects of a drug that disrupts microtubule polymerization on lymphoma cells in 3D co-culture systems. The interaction between lymphoma cells and neutrophil-like cells is explored to understand the mechanisms of cell survival and apoptosis.
Take a multi-well plate containing 3D spheroid monocultures of lymphoma cells, cancerous white blood cells, and co-cultures of lymphoma and neutrophil-like cells in a basement membrane matrix.
Add a test drug that binds to intracellular tubulin and disrupts microtubule polymerization, resulting in cellular apoptosis.
In the co-culture, direct lymphoma cell contact activates the neutrophil-like cells, inducing cytokine secretion.
These cytokines interact with lymphoma cell receptors, triggering downstream signaling cascades and upregulating anti-apoptotic protein expression, promoting cell survival.
Discard the media. Add a dissociation buffer to disrupt cellular adhesion.
Scrape the spheroids and incubate them under agitation. Transfer the suspension to tubes and continue agitation to form single-cell suspensions.
Introduce a fluorophore-conjugated antibody cocktail that labels lymphoma and neutrophil-like cells.
Add fluorophore-tagged annexin V and propidium iodide to label apoptotic and dead cells.
Using flow cytometry, detect increased lymphoma cell survival in the co-culture, indicating neutrophil-mediated protection against the 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.
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