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This assay was performed as instructed by the manufacturer Promega USA. Serial fluorescence readings were performed using a plate reader at 485 nm excitation and 520 nm emission. The SH-SY5Y cells were again used in this assay and the caspase inhibitors purchased from Calbiochem included Caspase-3 inhibitor II (Z-DEVD-FMK) Caspase-8 inhibitor II (Z-IETD-FMK) Caspase-9 inhibitor I (Z-LEHD-FMK) Caspase general inhibitor I (Z-VAD-FMK) negative control (Z-FA-FMK) and positive control doxorubicin HCL.
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In this study SH-SY5Y cell death induced by MSE appeared to be independent of p53 and p21 pathway. However the morphological features indicated apoptoticlike type of cell death. Based on these findings it was postulated that the mechanism of cell death of SH-SY5Y cells upon MSE treatment may not follow the common intrinsic pathway which requires the activation of tumour suppressor protein p53. Therefore the possible involvement of the caspase enzymes such as upstream caspases 8 and 9 which are involved in both intrinsic and extrinsic pathways and also the executioner caspases 3 and 7 were investigated. MSE mediated cell death was found to not involve any of the caspase cascades kratom dangers examined. Thus Kratom Laws kratom buy online paradise Colorado Witten this finding is consistent with the previous data which indicates that the apoptotic-like cell death seen for MSE treated SH-SY5Y cells is p53independent and caspase independent.
Another important microscopic observation was made after the final readings at the 1 hr time point which showed that all cells in the Control group appeared rounded and floating in the middle of the well. Similar observations were also noted for H202 MSE and MIT groups. Interestingly the majority of the cells which were treated with NAC Kratom Laws Colorado Witten prior to treatment with H202 appeared firmly attached to the bottom of the wells and had normal cell appearance. Brownish precipitations were also noted floating in all wells believed to be the hydrophobic fluorescent dye DCFH-DA.