Killing K562 Cells by Peripheral Blood Mononuclear Cells Exposed to Tobacco Product Preparations
Transcrição
Tumor cells activate effector immune cells to release cytokines and cytolytic granules containing perforin and granzymes. Perforin forms pores in the tumor cell membrane, facilitating granzyme entry into the cell and initiating apoptosis, causing cell death.
To analyze the cytolytic function of human peripheral blood mononuclear cells, PBMCs, following tobacco product preparation, TPP, exposure, take a multi-well plate with increasing TPP concentrations. A medium-containing well acts as the control.
Add PBMC suspension. TPPs' primary immunosuppressive agent, nicotine, binds to effector PBMCs' nicotinic acetylcholine receptors, downregulating the pro-inflammatory cytokine production.
Centrifuge. Remove the TPP-containing supernatant. Resuspend the PBMCs in media. Add fluorescently labeled K562 cells, human leukemic cells having an increased susceptibility to PBMC effector cell cytotoxicity, to the wells.
TPP-treated effector cells with suppressed immune responses exhibit reduced perforin expression and cytokine release, decreasing the PBMC's cytolytic ability.
Centrifuge. Resuspend the cells in a buffer followed by a cell-impermeable fluorescent dye. The dye enters the cells with compromised membranes and stains the DNA.
Using a flow cytometer, determine the proportion of dead and live K562 cells in the wells. A lower percentage of dead K562 cells in wells with higher TPP concentrations than the control indicates suppression of K562 cell killing by TPP-treated PBMCs.
