Calcein Acetoxymethyl Staining to Evaluate Natural Killer Cell-Mediated Cytotoxicity toward Cancer Cells
Published: November 30, 2023
Abstract
Source: Chava, S. et al., Measurement of Natural Killer Cell-Mediated Cytotoxicity and Migration in the Context of Hepatic Tumor Cells. J. Vis. Exp. (2020)
This video demonstrates an assay to measure the cytotoxicity of natural killer cells against cancer cells using the Calcein AM stain. The reduced number of fluorescent cancer cells following co-culture with natural killer cells indicates natural killer cell-mediated cytotoxicity toward cancer cells.
Protocol
1. Calcein AM Staining-based Microscopic Method for Measuring NK Cell-mediated Cytotoxicity Culture SK-HEP-1 cells to 70−80% confluency. Generate a single-cell suspension by first washing cells with 5 mL of 1x PBS followed by incubation with 1 mL of 0.25% trypsin-EDTA. Centrifuge SK-HEP-1 cells in a 1 mL sterile centrifuge tube at 160 x g for 3 min. Resuspend the pellet in 3 mL of serum-free DMEM. Add 1.5 µL of calcein AM solution (10 mM) to SK-HEP-1 ce…
Divulgaciones
The authors have nothing to disclose.
Materials
| Calcein AM dye | Sigma-Aldrich | 17783 | |
| DMEM | Gibco | 11965-092 | |
| Fetal Bovine Serum | Gibco | 26140079 | |
| Horse Serum | Gibco | 16050114 | |
| NK92MI cells | ATCC | CRL-2408 | |
| Phosphate Buffer Saline (PBS) | Sigma-Aldrich | P4417 | |
| SKHEP-1 Cells | ATCC | HTB-52 | |
| Trypsin EDTA solution | Gibco | 25200056 |
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Calcein Acetoxymethyl Staining to Evaluate Natural Killer Cell-Mediated Cytotoxicity toward Cancer Cells. J. Vis. Exp. (Pending Publication), e21783, doi: (2023).