Use of hexosaminidase in a cytotocicity assay

Friday, November 07, 2003

Description
A simple enzymatic method to estimate cell death/viability of adherent cells
Original reference: Landegren, U., 1984. Measurement of cell numbers by means of the endogenous enzyme hexosaminidase. Applications to detection of lymphokines and cell surface antigens. J. Immunol. Methods 67, pp. 379388. Abstract

Procedure
Cells are layered in 96-well plate and incubated using your potentially cytotoxic condition (CD8 T cells, cytotoxic product, etc...)
The cells are washed with warm PBS to remove dead cells
The next step is the incubation with 50 microL hexaminidase substrate (NPAGA) (duration depends on your condition, this needs to be established for your cells)
The plate is incubated at 37 (beware of evaporation)
100 microL of stopping buffer are then added
OD is measured at 405 nm

Recipes
NPAGA: 3.75 mM p-nitrophenyl N-acetyl-beta-D-glucosaminide (Sigma, Saint Louis, MO)) dissolved in 0.1 M citrate buffer and mixed just before the experiment with an equal volume of 0.5% Triton X-100 in water
stopping buffer: 50 mM glycine buffer, pH 10.4, with 5 mM EDTA

Supplies

Tips
You have to make sure that you cell line has this endogenous hexaminidase activity
Coloration appears only after addition of the stopping buffer

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Use of hexosaminidase in a cytotocicity assay