24-well plate spectrophotometric assay for preliminary screening of metabolic activity.
A 90-min assay using 24-well plates was developed for screening overall metabolic effects of toxic chemicals by measuring extracellular acidification rate of mammalian cells. During the 90-min test, the pH of each well of a 24-well plate is monitored by measuring the absorbance of phenol red using a spectrophotometric plate reader. The acidification rate is then calculated from the change in pH divided by the time interval and cell density, which is also estimated non-invasively by spectroscopy. The assay was verified by testing the effects of five well-characterized chemical toxins on fibroblast cell cultures. As expected, the responses of the fibroblasts were dependent on the dose and type of toxin. They also corresponded with the established mechanisms of the toxins and with measured lactate production rates. The set-up is simple, inexpensive, and amenable to being automated. The method is easy to perform and rapid. Uses include screening compounds for gross metabolic effects in mammalian cell lines, determining preliminary metabolic dose-response curves for guiding further research, and designing and optimizing media for in vitro systems utilizing cell cultures.
Yang
Yuansheng
Y
Department of Chemical Engineering, Vanderbilt University, Nashville, TN 37235-1604, USA.
Balcarcel
R Robert
RR
D019297:2,4-Dinitrophenol / Q000032:analysis; D000818:Animals; D000968:Antimycin A / Q000032:analysis; D002460:Cell Line; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D015386:Hazardous Substances / Q000032:analysis* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D051379:Mice; D010072:Oxamic Acid / Q000032:analysis; D015203:Reproducibility of Results; D012402:Rotenone / Q000032:analysis; D012969:Sodium Fluoride / Q000032:analysis; D013053:Spectrophotometry / Q000379:methods; D013997:Time Factors
D015386:Hazardous Substances; D012402:Rotenone; D000968:Antimycin A; D012969:Sodium Fluoride; D019297:2,4-Dinitrophenol; D010072:Oxamic Acid