DEVELOPMENT OF A PURIFICATION METHOD OF PURE PRIMARY LYMPHOCYTES FOR CELL VIABILITY ASSAYS

The maintenance of pure primary lymphocytes culture for long periods may be difficult because of its inability to divide continuously. In addition, lymphocytes separation methods such as Ficoll-Paque, RBC lysis and immunomagnetic microbeads separation may have some affect on cell viability. The objective of this study is to determine various types of lymphocytes purification methods, in order to prolong primary lymphocytes culture to 72 hours. The second objective is to use these primary lymphocytes as targets for quantitative and qualitative cell viability assays when analysing the action of toxins isolated from natural products. Human blood was drawn and purified by using Ficoll-Paque, RBC lysis or immunomagnetic separation column method in various combinations. The purified lymphocytes were also grown with and without the growth enhancement factor, concanavalin-A. Cell viability assays were carried out for 72 hours at 24 hours interval. The lymphocytes purified using RBC lysis method, with or without concanavalin-A can prolong 100% cell viability for 72 hours whilst lymphocytes purified using Ficoll-Paque and supplemented with concanavalin-A showed an increase in cell viability of over 250% at 72 hours incubation. It was observed only lymphocytes purified using Ficoll-Paque followed by the immunomagnetic microbeads separation method and supplemented with concanavalin-A showed overall cell viability increase, reaching 300% at 72 hours incubation. This method was a reliable model to test the cytotoxicity of the Bacillus thuringiensis parasporal inclusion, suggesting that the method achieves the objectives of the study.