|Kehrli Jr, Marcus|
Submitted to: Great Lakes International Imaging and Flow Cytometry Association
Publication Type: Abstract Only
Publication Acceptance Date: 9/30/2005
Publication Date: 9/30/2005
Citation: Kimura, K., Lager, K.M., Kehrli, Jr., M.E., Roth, J.A., Richt, J.A. 2005. Assay for proliferation of porcine PBMC with Oregon Green 488 and its application for evaluation of T cell responsiveness to swine influenza virus [abstract]. Great Lakes International Imaging and Flow Cytometry Association. Paper No. 10. p. 22.
Technical Abstract: Cell proliferation assay is a useful method to analyze cell-mediated immunity to microbial antigens. In order to define T cell responsiveness to virus antigens in pigs, we have developed flow cytometric assays for porcine PBMC proliferation together with assays for intracellular IFN-g, and cell surface expression of CD25. Carboxy-fluorescein diacetate, succinimidyl ester (CFSE) is widely used for lymphocyte proliferation assays in human and rodents, but the application for swine research is not reported. CFSE is toxic to PBMC thus maximum usable concentration is 3 uM. Using this concentration fluorescent intensity fades during a 5-day incubation period, which results in fluorescence barely distinguishable from background of non-stained cells. In order to avoid these problems, we have applied a newly available dye, Oregon Green 488, which has very similar characteristics to CFSE. With the same staining procedure and concentration as CFSE, Oregon Green has higher fluorescent intensity than CFSE, thus we can reduce background by reducing photomultiplier tube voltage, and fluorescence is clearly distinguishable from background, even after 7 days of incubation. We have compared cell proliferation assays using Oregon green with BrdU incorporation, and found the assays were very well correlated (r = 0.7307). We have applied this technique together with intracellular IFN-g, and CD25 cell surface expression assays in order to determine recall response of PBMC to the swine influenza virus (H3N1) antigen after infection. All three parameters showed increase after in vitro antigen stimulation compared to sham stimulation in pigs previously infected with swine influenza virus but not in non-infected control pigs (% proliferation: 8.79 +/- 1.78% vs. -0.64 +/- 1.39%. % CD25 expression: 6.58 +/- 1.88% vs. 0.00 +/- 2.23%. % IFN-g positive (with Brefeldin A for 4 h): 2.40 +/- 0.84% vs. -0.13 +/- 0.42%). Cell surface expression of CD4 and CD8 was analyzed simultaneously and results showed that about 30 to 40 % of activated cells were CD4+ cells and about 20 % of activated cells were CD8+ cells. Although there were only 2 pigs in each group (non-infected and infected), the results were consistent indicating that these techniques are useful to evaluate cell-mediated immunity in pigs to viral antigens.