The Application of MTT Colorimetric Assay to Measurement of Blastogenesis in Canine Peripheral Lymphocytes

Abstract

Blastogenesis of canine peripheral lymphocytes was measured by the MTT (3-(4, 5-dimethylthiazole-2-yl)-2, 5-diphenyl tetrazolium bromide) colorimetric assay. MTT is cleaved only by living cells and formazan is produced. This process needs active mitochondria, and even freshly dead cells do not cleave MTT. Canine peripheral lymphocytes prepared by Ficoll-Conray gradient were cultured with lectins (PHA and Con A) for 96 h at 37°C under 5% CO₂ using the microplate system. At the end of the culture, the MTT reagent and HC1-isopropanol were added to the culture, and then, the absorbance of each culture was measured by a microplate reader using a test wavelength of 570 nm, and a reference wavelength of655 nm. The mean value of SI (stimulation index) of canine lymphocytes from 14 normal dogswas 183±130 in PHA stimulated culture and 131±82 in Con A stimulated culture. The administration of predonisolone (5 mg/kg) to 3 normal dogs at 6 times over 3 days depressed SI from 69±31 to15±9 in PHA, from 70±14 to 6±4 in Con A. In conclusion, the MTT assay provides an efficient evaluation method for lymphocyte blastogenesis in dog in vitro.