Wilson Lewis Mandala1
Cell-mediated responses to immunological stimuli are often localized in inflammatory sites and involve a number of cell types. These responses can be functionally characterized at the single-cell level on the basis of the types of cytokines expressed. The ability to measure antigen-specific cell responses at the single cell level is an important tool with a wide range of potential applications ranging from studies of disease pathogenesis to the evaluation of vaccines. A number of experiments were performed in this study in order to establish the optimal conditions for stimulating in vitro cytokine production by lymphocytes and monocytes in blood samples collected from healthy adult Malawian participants and the optimal staining conditions for various cytokines and cell types. Different stimulation methods and conditions, different culture tubes and incubators and different antibody labelling conditions were assessed in order to establish optimal conditions.
The use PMA plus Ionomycin produced highest cytokine-producing T cells whereas LPS was a better stimulant for cytokine producing monocytes. Stimulation of whole blood for five hours was optimal for cytokine detection in T cells whereas four hours was optimal for monocytes. BFA was found to be a better Golgi blocker than Monensin and that the use of 15ml Falcon-type polypropylene tubes while stationary resulted in the detection of the highest proportion of cytokine-producing cells. T cells were found to be producers of mainly TNF-α, IFN- and IL-2 whereas Monocytes were mainly producing TNF-α and IL-6. 2µl of anti-CD3-PerCP, 2µl of anti-CD14-APC and 4µl of anti-cytokine-PE resulted in the best results. The highest cytokine production monocytes were detected when 2ml of FACS Lysing solution was used compared to the other volumes. These optimal conditions are essential in determination of proportion of cytokine-producing cells using ICS.