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MSE fractionation was performed using solid phase extraction (SPE) method using polymeric strong cationic exchange sorbent which was a kind gift from Phenomenex Company (U. Buy Kratom Online Australia the extraction method was a modified procedure of the SPE method also obtained from Phenomenex Company. MSE was weighed and dissolved with 3 ml absolute methanol and 21 ml 20% formic acid.

Active caspase 9 will activate the downstream caspases 3 6 and 7 for cells to execute apoptosis (Ghobrial et al 2005) (refer to fig. Final execution:

Caspases pathway As described above in the two main pathways caspases which belong to cycteine proteases family play important roles in the initiating and executing the final apoptosis events. The

well known caspases which are involved in apoptosis are initiator or upstream caspases 8 9 and 10 and executor or downstream caspases 3 6 and 7.

Fresh medium was added to inactivate the trypsinisation process and for detachment of cells. The suspended cells were split 1:3 every 3-4 days. Cells were grown to subconfluency and harvested as described for HepG2 cells. C in a water bath (1 min) in order to minimises the toxic effects exerted by the cryoprotectantDMSO on thawed cells. The content of the vial (1.

The assessment of p53 levels and its kratom pills or extract target gene p21 which are highly associated with apoptotic cell death can also be investigated using many in vitro approach such as immunoblotting (Western blot) fluorescence image cytometry etc (Mckenzie et al 1999). The generation of ROS in mediating the cell death should also be a major concern in investigating the in vitro assessment of cell death as ROS is a major indicator for mitochondrial dysfunction which in turn kratom powder smell could activate many forms of programmed cell death (Tan et al 1998) and a ommon method to measure the ROS generation in live cells is using the 27-dichlorofluorescein dye (DCFH) (Esposti 2002). Justification Objectives and Hypothesis 1.

IC50 values (Inhibition concentration that caused 50% cell death) of 24 hr treatment with MSE and MIT treated cell lines. The values were interpolated from

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percentage dead cells curves obtained from the Trypan blue kratom extract for pain exclusion experiments. MIT (Molar) 7. MSE and MIT. From these estimates it appears that the SH-SY5Y cells are the most sensitive of those examined to the cytotoxic and possibly cytostatic effect of MSE.