The Effect of Phytohaemagglutinin on Measurement and Evaluation of Lymphocytes using MTT Assay

Abstract

MTT assay is a technique used to detect immunological property of cells. However,

the limitation of this technique is the aggregation of cells in experiment wells. The PBMC aggregation,  which  is found  after stimulation with PHA,  affected  absorbance measurement. To reduce PBMC aggregation, the present study investigated the optimal concentration and incubation time of PHA to induce PBMC proliferation under chelating agent (EDTA) supplemented medium. Peripheral blood samples were provided for PBMC isolation using Ficoll-Hypaque density gradient centrifugation. The PBMC was induced proliferation under various concentrations of PHA with a supplementation of EDTA. The reactions were measured by MTT assay. The results showed that PHA at a concentration of 1  µg/mL presented  a low

amount of  PBMC aggregations  resulting  in significantly  high absorbance value, whereas,  a

concentration of 5 µg/mL PHA showed high aggregation amount of PBMC and a low absorbance value as compared to that at 1 µg/mL PHA. EDTA was found to be an effective supplement for reduction of aggregation in PBMC cultures and it was concluded that optimal concentration of PHA and supplementation of EDTA can reduce PBMC aggregation in reaction for MTT assay.