Adaptation of the diphenylamine (DPA) assay to a 96-well plate tissue culture format and comparison with the MTT assay.
A simple spectrophotometric method for measuring DNA in proliferating cells is described. The method is an adaptation of the widely used diphenylamine (DPA) reaction to examine DNA in cells grown in a 96-well plate. This assay was capable of detecting as little as 10 ng DNA and could be used to measure DNA in stored as well as viable tissue samples. The DPA assay paralleled the MTT (3-[4,5-dimethyl-thiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay of mitochondrial reductase in A549 cells, a human lung cancer cell line; in EMT6 cells, a mouse breast cancer cell line; and in a primary cell culture of neonatal rat astrocytes. Over several days of proliferative growth in tissue culture, the ratio of MTT to DPA remained constant. Since the DPA assay and MTT assay measured different parameters of the same cells, they could be employed as complementary spectrophotometric assessments of cell growth on a 96-well plates using the same automated enzyme-linked immunosorbent assay plate reader.
Natarajan
N
N
Veterans Affairs, Lakeside Medical Center, Chicago, IL.
Shambaugh
G E
GE
Elseth
K M
KM
Haines
G K
GK
Radosevich
J A
JA
D000818:Animals; D002455:Cell Division; D004247:DNA / Q000032:analysis*; D004159:Diphenylamine; D051381:Rats; D017207:Rats, Sprague-Dawley; D013053:Spectrophotometry; D013778:Tetrazolium Salts; D013844:Thiazoles; D014407:Tumor Cells, Cultured
D013778:Tetrazolium Salts; D013844:Thiazoles; D004247:DNA; D004159:Diphenylamine; C022616:thiazolyl blue