CIK cells harvested between days 14 and 21 were challenged against tumor targets using a standard calcein-acetoxymethyl (Sigma-Aldrich) release assay. Specifically, 10 6 tumor cells were labeled with 3.5 mM calcein-acetoxymethyl in complete RPMI medium for 30 minutes at 37°C and then washed with Dulbecco's Phosphate-Buffered Saline (Sigma Aldrich). Tumor cells were then plated in 96-well U-bottom plates and incubated with CIK cells at the indicated E:T ratios at 37°C. Maximum and spontaneous release was obtained by incubating target cells with 3% Triton X-100 (Sigma Aldrich) or complete RPMI medium. After 4-hour incubation, the plates were centrifuged, and 100 mL of supernatant collected from each well was transferred into an OptiPlate-96 black plate (PerkinElmer) to measure the released fluorescence using the VICTOR multilabel plate reader (PerkinElmer). The results of the cytotoxicity assay are expressed as specific lysis, which is calculated as follows: % specific lysis = ([experimental release À spontaneous release] / [maximum release À spontaneous release]) £ 100.
Calcein acetoxymethyl
Manufactured by Merck Group
Calcein-acetoxymethyl is a fluorescent dye used in cell biology and biochemistry applications. It serves as a calcium indicator, allowing researchers to monitor intracellular calcium levels within living cells.
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2 protocols using calcein acetoxymethyl
1
Cytotoxicity Assay of CIK Cells
2
Monocyte Adhesion to Vascular Cells
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THP-1 cells were labeled with 2.5 μmol/l calcein acetoxymethyl (Sigma-Aldrich) for 30 min. The labeled cells were washed with PBS 3 times to remove the residual calcein acetoxymethyl. calcein acetoxymethyl-labeled THP-1 cells (1 × 105) were added to a confluent monolayer of hASMCs and hAECs for 1 h, and then washed with PBS 3 times. Adherent monocytes were quantified in 6 randomly selected microscopic fields (×20) using the ImageJ software.
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