Apoptosis in mouse J774 macrophages : a methodological study

• Main Author: Imam, Hasan
• Format: Others
• Language: English
• Published: Linköpings universitet, Institutionen för klinisk och experimentell medicin 2009
• Online Access: http://urn.kb.se/resolve?urn=urn:nbn:se:liu:diva-73881

The approach of studying apoptosis induced by UV ray and actinomycin treatment was done to find a suitable positive control for that apoptosis study, with murine macrophage (J774 cell line). This scientific project was done in order to develop a method with this cell line in our laboratory condition for further apoptosis study in the presence of M.tuberculosis bacilli. It is well known that Mycobacterium tuberculosis primarily targets the macrophages and use them as a critical reservoir for the infection in the lung. Macrophages show several antimicrobial activities in order to limit the spread of intracellular infection during an attack by tuberculosis bacilli and apoptosis is one of the innate defense mechanism. Our findings were important because it gives the necessary information’s regarding the possible sites where care is to be taken during working with this cell line (J774). We investigated the effect of UV exposure and actinomycin D treatment to find the suitable positive control for the study of apoptosis in J774 cells. Moreover, we studied effect of yeast particles on apoptosis and the phosphorylation of p38MAPK at two different time intervals in J774 cells. In order to quantify the percentage of apoptotic cells, we stained the cells by FITCAnnexin V, Propidium Iodide, and TMRE and evaluated the results by flow-cytometry. Additionally, the phosphorylation of p38MAPK was assessed by western blot. The results indicated steady increase in apoptosis, measured by both annexin-V and TMRE 18 hours after UV exposure. Also we investigated phosphorylated p38MAPK after 18 hours from UV exposure (result not given) but no band was found. Actinomycin on the other hand, had no effect on the number of apoptotic cells and further study is needed to optimize its concentration. Non opsonised yeast did not induce any increase in annexin-V staining compared to the control, but did surprisingly induce a decrease in the mitochondrial membrane potential after 6hours, an effect that was diminished after 18 hours, here also we didn’t find any band of that phosphorylated p38MAPK during immunoblotting. Similar data were found with serum opsonised yeast particles. Maybe the beta-glucan of yeast particle was involved here by producing reactive oxygen species via NADPH oxidase and consequent mitochondrial membrane potential loss but we couldn’t draw any conclusion from our result about apoptosis occurring.