Encyclopedia of Experiments: Immunology
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Transcript
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.
