Determination of the number of endothelial cells in culture using an acid phosphatase assay

✍ Scribed by Daniel T. Connolly; Mary Beth Knight; Nikos K. Harakas; Arthur J. Wittwer; Joseph Feder

• Publisher: Elsevier Science
• Year: 1986
• Tongue: English
• Weight: 361 KB
• Volume: 152
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1016/0003-2697(86)90131-4

✦ Synopsis

A simple assay is described in which small numbers of endothelial cells in culture can be determined by measuring acid phosphatase activity. After removal of the growth medium from cells grown in 96-well culture plates, the cells are lysed in buffer containing the detergent Triton X-100 and the phosphatase substratep-nitrophenyl phosphate. After 2 h at 37"C, the reaction is stopped with sodium hydroxide, and color development is determined using a rapid multiwell plate reader. The assay detects 100 to 10,000 cells per well. The assay has been used to determine growth curves for endothelial cells in the presence and absence of endothelial cell growth factor from bovine hypothalamus and to monitor fractions during purification of the growth factor. Minor modifications in the assay allow it to be fully automated. 0 1986 Academic press, hc.