The CytoTox 96® Non-Radioactive Cytotoxicity Assay is designed for
use with a target cell type and an effector cell type to measure cell-mediated
cytotoxicity. Can it be used with a single cell type to determine the cytotoxicity of a
chemical compound or other treatment?
Yes. This system (Cat.#
G1780) can be adapted for use with a single cell type quite easily. Cells treated with
cytotoxic compounds will die and release LDH into the medium. The CytoTox 96®
System measures the amount of LDH in the culture medium and indicates the number of cells
that have died or that have compromised cell membranes.
The protocol for use of this system (#TB163) with a
single cell type is essentially the same as for use with a target/effector cell assay.
The following controls are suggested when using this system for a single cell type
assay:
- Cell spontaneous release control (controls for any spontaneous cell death).
- Test compound/vehicle control (controls for any potential effect the test compound may
have on the tetrazolium redox reaction).
- Maximum LDH release control. Obtain by detergent lysis of cells (controls for maximal
possible LDH).
- Volume correction control (controls for volume increase with maximum release).
Simplified Protocol:
- Determine the optimum number of cells needed as described in the CytoTox 96® Non-Radioactive Cytotoxicity
Assay Technical Bulletin #TB163. There is no need to use V-bottom or round-bottom
plates.
- Plate cells in a 96 well plate along with the controls described above.
- Add the test compound.
- Incubate the plate for an appropriate length of time. Forty-five minutes prior to
harvesting supernatants, add 10µl of Lysis Solution (10X) for every 100µl of target
cells to the wells containing the target cell maximum LDH release control. Add the same
volume of lysis solution to the volume correction control.
Note: If the target cells are not lysed completely (as determined by
microscopy), add an additional 5µl of Lysis Solution.
- Remove 50µl aliquots from each well and transfer to a fresh 96 well plate.
- Add the substrate solution, incubate, stop and read the absorbance as described in the
Technical Bulletin.
Cytotoxicity, as % maximum release, can be expressed as:
| (Absorbanceexperimental*
- Absorbancespontaneous) x 100 |
| (Maximum release - Volume correction control) |
*Includes vehicle control for percent release due to vehicle alone.
CytoTox 96 is a trademark of Promega Corporation and is registered with
the U.S. Patent and Trademark Office.
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