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Cytotoxicity of Extract of Malaysian Mitragyna Speciosa Korth and Its Dominant Alkaloid Mitragynine

Cytotoxicity of Extract of Malaysian Mitragyna Speciosa Korth and Its Dominant Alkaloid Mitragynine

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PHD thesis on the safety of kratom
PHD thesis on the safety of kratom

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Published by: spangled101 on Jan 03, 2011
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02/12/2013

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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. One hundred thousand cells/well in 6 well
plates were cultured overnight. The cells were then treated with 10 µM of each
inhibitor 30 minutes prior to adding the MSE. The treated cultures were incubated
at 37ºC (5% CO2) for 48 hr time period. After incubation, the cells were
harvested and trypsinised as described in chapter 2 section 2.2.2. The cell pellets
were then prepared for flow cytometry analysis using PI staining as described in
chapter 4, section 4.2.4. The cells stained with PI were analysed using BD
FacsCalibur flow cytometer. PI was excited at 488 nm and 620 nm emissions. Ten
thousand cells were analysed by CellQuest Pro software and the subG1 population
representing apoptotic cells were gated manually.

5.2.3.4 Reactive oxygen species (ROS) analysis in SH-SY5Y cells treated with
MSE and MIT

ROS generation assay was carried out using SH-SY5Y cells by using a
fluorescent dye 2,7-dichlorofluorescein diacetate (DCFH-DA). Principally, this
dye diffuses through the cell membrane and is hydrolysed enzymatically by
intracellular esterases to form monofluorescent dichlorofluorescein (DCFH) in the
presence of ROS. The intensity of the fluorescence is therefore, proportional to
the levels of intracellular ROS (Galvano et al, 2002).

A fluorescence-based method to measure ROS generation in live cells was a
modification of the procedure described by Esposti and McLennan (1998).
Briefly, SH-SY5Y cells (5 x 105

cells/well) in 6 well plates were cultured
overnight. Then the medium was aspirated and washed with PBS to remove all

121

traces of albumin present in the medium. This is to ensure that the free-radical
quencher albumin present in the serum used as a media supplement is removed as
it interferes with the quantitative analysis of ROS (Esposti, 2002). PBS (2 ml) was
added per well followed by addition of freshly prepared DCFH-DA dye dissolved
in DMSO (100 µM) under subdued lighting. Anti-oxidant, N-acetyl-L-cysteine
(NAC) (5mM) was also added to appropriate wells. Fluorescent was measured
using a plate reader with 485 nm excitation and 530 nm emission. After 30
minutes, cells in each well were treated with H202, MSE and MIT and the
fluorescent readings were continually read at 10 min intervals for up to 1 hr
period. This preliminary assay was performed to establish the working conditions
for the assay.

Further optimisation of the ROS assay was performed using SH-SY5Y cells (2 x
103

cells/well) in 24-well plates cultured overnight. As described earlier, the
cultured medium was aspirated and fresh PBS (1 ml) was added to each well. The
fluorescent dye, DCFH-DA (100 µM) was then added to the wells under subdued
lighting and NAC was also added to appropriate wells. The cells were incubated
at 37ºC (5% CO2) for 30 minutes. As the addition of DCFH-DA dye led to
precipitation as seen in the preliminary experiment, after 30 min the cultured
solutions were aspirated and fresh PBS (1 ml) was added to each well prior to
adding the test compounds (H202, MSE and MIT). The fluorescence readings were
then taken every 10 minutes interval up to 1 hr as described earlier.

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