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 buy kratom west palm beach doxorubicin HCL. M of each inhibitor 30 Kratom Natural Drug minutes prior to adding the MSE. C (5% CO2) for 48 hr time period. Kratom Natural Drug after incubation the cells were harvested and trypsinised as described in chapter 2 section 2. The cell pellets were then prepared for flow cytometry analysis using PI staining as Kratom Natural Drug described in chapter 4 section 4.
As shown in fig. A there was a non-significant difference noted for caspase 3 and 7 activities for MSE treated
cells compared to control groups at 4 hr incubation time point. For MIT treated cells (Fig. M showed significant differences compared to control group for all fluorometric readings. For 18 hr incubation time period (Fig.
BCA) protein assay kit from Pierce (Rockford IL). Primary antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz CA) and Oncogene Research Products (Darmstadt Germany) and secondary antibodies were from Sigma-Aldrich (U. Santa Cruz Biotechnology (Santa Cruz CA).
Cell cycle arrest which is known to be highly associated with cytotoxicity was seen in the present study and SH-SY5Y cell again was the most vulnerable cell line to the MSE and MIT effects. M phases for the HEK 293 cells. This phenomenon was found in all cell lines examined and indicates that more PI dye was taken up by the cells thus an increase in the DNA staining intensity.
The same outcome was also noted for caspase 9 assay which was performed using the same cell Kratom kratom bali kratom vs vendor usa Natural Drug lysates (Fig. C and D). At the 24 hr time point of both caspase assays (Fig.
C for 10 min. The reaction was terminated with stop solutions provided with the kit. The plate was read using a fluorescent plate reader with an excitation wavelength of 560 nm and emission wavelength of 590 nm.
The vendor said he had the leaves completely boiled i. At the first I found the taste disgustingly bitter but subsequently I had no problem swallowing it. I consumed it over a 2 week period of about 1.
CYP 2E1 inducers for example alcohol. If time had permitted the role of metabolism in activating MSE and MIT would have been an important area to pursue. As part of a toxicological assessment genotoxic potential of a compound is important to characterise. A genotoxic agent is capable of causing DNA damage and if repair is unsuccessful it can lead to further major problems such as carcinogenesis.
Among these ROS H2O2 is the most stable and abundant (Esposti 2002) and has a relatively long half-life(Lu et al 2007). In this part of the study morphological features of the cells treated with MSE were cytologically examined using Wright-Giemsa or Rapi-Diff staining. Flow cytometry analysis using Annexin V conjugate assays were employed in order to distinguish the mode of cell death upon treatment with MSE and MIT.