Use of hexosaminidase in a cytotocicity assay
时间:2005-07-18 00:00 来源:Scienceboard.net 作者:admin 点击:次
| 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 (责任编辑：泉水) |