This study investigates the cytotoxic effects of natural killer (NK) cells on cancer cells using a multi-well plate assay. The methodology involves measuring lactate dehydrogenase (LDH) release as an indicator of cancer cell lysis.
Take a multi-well plate containing natural killer, or NK, cells and cancer cells. Control wells contain only cancer cells.
Centrifuge to facilitate cellular contact.
Incubate. Activating receptors on NK cells bind to cancer cell surface ligands, triggering NK cells to release granules containing cytotoxic molecules.
These molecules create cancer cell membrane pores and induce apoptosis releasing intracellular contents, including lactate dehydrogenase, or LDH.
Add a detergent to lyse the cancer cells in control wells for maximum LDH release.
Centrifuge and transfer a portion of LDH-containing supernatants to an assay plate.
Add the assay reagent and incubate.
LDH converts lactate to pyruvate reducing NAD+. Diaphorase uses NADH to reduce tetrazolium salt, forming a red-colored formazan product.
Add an acidic solution to stop the enzymatic reaction.
Use a microplate reader to measure the formazan absorbance in the wells.
The formazan intensity is proportional to the number of lysed cancer cells in the test well, indicating NK cell cytotoxicity against cancer cells.
First, obtain a round-bottom culture-treated 96-well plate, and set it up as outlined in table 1 of the text protocol. Centrifuge the assay plate at 250 times g for 4 minutes to be certain that the effector and target cells are in contact. Next, incubate the plate in a humidified chamber at 37 degrees Celsius with 5% carbon dioxide for 5 hours.
45 minutes prior to harvesting the supernatants, add 10 microliters of 10x lysis solution to the target cell maximum LDH release wells, and return the plate to the humidified chamber. When the incubation is complete, centrifuge the plate at 250 times g for 4 minutes. Then, use a multichannel pipettor to transfer 50-microliter aliquots from every well to a fresh 96-well flat-bottom assay plate.
Add 50 microliters of assay reagent to each well of the assay plate. Cover the plate with foil to protect it from light, and incubate at room temperature for 30 minutes. After this, add 50 microliters of stop solution to each well. Make sure to read the plate within 1 hour of adding the stop solution and record the absorbance at 490 nanometers.
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