Leah Borkan Paper II
In this paper, the hypotheses being investigated were whether influenza A virus was capable of inducing apoptosis in mast cells, and if so determine if the virus was using this intentionally to its benefit and which pathways were utilized. The methods used included viral infection of cell cultures, hemagglutination assay, real-time quantitative PCR, plaque assay, transmission electron microscopy, TUNEL assay, flow cytometric analysis of apoptosis, western blot, subcellular fractionation extraction, inhibition of apoptosis using specific inhibitors, and cytokine and chemokine quantification. The replication efficiency of H1N1, H5N1, and H7N2 in P815 mast cells was analyzed in figure 1. A HA assay examined red blood cells’ contact with IAV’s protein envelope, which resulted in clumping. The standard plaque assay was used to determine viral titers for times post infection. Lastly, PCR was used to quantify the amount of viral genes present at certain times post infection. The results of these experiments showed that the replication of the virus in mast cells were more productive in H1N1 and H5N1 versus H7N2. This not only proved IAV’s replication capability in mast cells, but also suggests …show more content…
Intrinsic apoptotic marker caspase 9 and extrinsic apoptotic marker caspase 8 were cleaved and activated to be analyzed. Members of the Bcl-2 family, caspase 3, and Apad-1 were also analyzed. The results showed that in the infected cells, pro-caspase 8 degradation remained minimal after infection and was barely more apparent than the mock cells. 12 hours post infection Caspase 9 activated cleaved fragments were notable, and at 24 hours post infection were considerably greater. These results point towards the mitochondria-mediated intrinsic apoptosis being the main pathway involved in the IAV infection of mast
In this paper, the hypotheses being investigated were whether influenza A virus was capable of inducing apoptosis in mast cells, and if so determine if the virus was using this intentionally to its benefit and which pathways were utilized. The methods used included viral infection of cell cultures, hemagglutination assay, real-time quantitative PCR, plaque assay, transmission electron microscopy, TUNEL assay, flow cytometric analysis of apoptosis, western blot, subcellular fractionation extraction, inhibition of apoptosis using specific inhibitors, and cytokine and chemokine quantification. The replication efficiency of H1N1, H5N1, and H7N2 in P815 mast cells was analyzed in figure 1. A HA assay examined red blood cells’ contact with IAV’s protein envelope, which resulted in clumping. The standard plaque assay was used to determine viral titers for times post infection. Lastly, PCR was used to quantify the amount of viral genes present at certain times post infection. The results of these experiments showed that the replication of the virus in mast cells were more productive in H1N1 and H5N1 versus H7N2. This not only proved IAV’s replication capability in mast cells, but also suggests …show more content…
Intrinsic apoptotic marker caspase 9 and extrinsic apoptotic marker caspase 8 were cleaved and activated to be analyzed. Members of the Bcl-2 family, caspase 3, and Apad-1 were also analyzed. The results showed that in the infected cells, pro-caspase 8 degradation remained minimal after infection and was barely more apparent than the mock cells. 12 hours post infection Caspase 9 activated cleaved fragments were notable, and at 24 hours post infection were considerably greater. These results point towards the mitochondria-mediated intrinsic apoptosis being the main pathway involved in the IAV infection of mast