Calcein release assay to detect cytotoxic T-lymphocyte activity assay

Summary

The source of this experiment is the official website of the Fourth Military Medical University

Operation method

Calcein release assay to detect cytotoxic T-lymphocyte activity assay

Principle

Calcein acetoxymethy1 ester (Calcein-AM) is a cytoplasmic fluorescent marker, which is non-fluorescent itself, and the water-soluble green fluorescent substance catalyzed by the intracellular esterase is not easily permeated out of the cell after penetration. The target cells are labeled with it and co-cultured with effector cells, then Fluoro-Quench reagent (a main component of calf hemoglobin chelated with Ca2+, also containing ethidium bromide reagent, which is non-toxic to the cells, and can not enter into the living cells but may enter into the dead cells whose membranes have been broken) is added to quench the fluorescence of the culture medium, and the fluorescence intensity in the living cells is quantitatively measured on a plate fluorescence scanner, and the fluorescence intensity in the control wells (representing 100% survival of cells) is compared with that of the target cells. control wells (representing 100% cell survival). The percentage of target cells killed by effector cells can be calculated. This method is simple and reliable, no need to collect and transfer culture supernatant, high reproducibility, low variability, the measurement data are automatically input into the computer, suitable for large quantities of measurement; CTL, LAK and NK cell activity can be measured, but also in the optical microscope or fluorescence microscope to observe the cells directly.

Move

1. Labeling of target cellsLabeling was performed with 5 μmol/L Calcein AM 37℃ water bath for 30 min.2. Effector-target cell effectsKilling experiments were performed in round-bottom 96-well culture plates. Gradiently diluted effector cells and labeled target cells were added at E/T ratios of 50/1, 25/1, 12.5/1, 6.25/1, 3.13/1, 1.56/1, each l00 μl/well, with 3 replicate wells for each ratio. At the same time, 3 replicate wells were set up for the spontaneous release group and 3 replicate wells for the complete release group. The effector-target cell interaction 96-well culture plate was incubated at 37℃ 5% CO2 incubator for 4h, then centrifuged at 3000r/min for 5min, and the supernatant was transferred into new wells.3. Fluorescence detection

The fluorescence value (FI) of the supernatant was detected using a fluorometer with an excitation light of 485 nm and an emission light of 538 nm.4. Calculation of resultsKill rate (%) = ■ x 100%

For more product details, please visit Aladdin Scientific website.

https://www.aladdinsci.com/