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PBS followed by centrifugation (1200 r. Cells were re-suspended in Annexin-binding buffer (10mM HEPES 150 mM NaCl and 2. M CaCl2 at pH 7. Best Otc Drug Opiate Withdrawal the cells were then incubated on ice for 5 minutes until data acquisition with a Becton Dickinson FACSCalibur flow cytometer using CellQuest Pro software. Annexin V conjugate was measured at 650 nm excitation and 665 nm emission and 7-AAD at 488 nm

excitation and 620 nm emission.

The bacterial tryptophan reverse mutation assay with Escherichia coli WP2. Rapid colorimteric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. Immunol Methods 65: 5563. Strategy for genotoxicity testing and stratification of genotoxicity test results- report on initial activities of the IWGT Expert Group. Genetic Toxicology and Environmental Mutagenesis 540 177-181. Kratom Murray A.

CYP 2E1 is an important xenobiotic metabolising enzymes for human and rodents which is expressed in the liver. CYP 2E1 can metabolise various substrates including paracetamol fluoxetin alcohol caffeine and many others (Tanaka et al 2000). CYP 2E1 inducers for example alcohol.

Tris 2 g SDS in 500 ml distilled water pH 8. Sources and dilutions of primary and secondary antibodies for p53 and p21 protein used for the immunoblot assay. The DNA profiles of three different cell lines (HEK 293 MCL-5 and SH-SY5Y cells) treated with MSE and MIT were assessed using nucleic acid staining with PI and analysed with BD FacsCalibur flow cytometer in the Centre for Molecular Microbiology and Infection (CMMI) core facility unit Flowers Building South Kensington Campus.

MSE were observed and mechanisms other than direct genotoxicity per se can lead to false positive results which are related to cytotoxicity and not genotoxicity such as events associated with apoptosis etc (ICH 1995). Such events are likely to happen once a certain concentration threshold is reached for a toxic compound. For instance in figure 2. A in the Best Otc Drug Opiate kratom 101 hawick Withdrawal previous chapter (section 2. MSE in the presence of S9 Best buy kratom online amazon Otc Drug Opiate Withdrawal reduced the colony formation to less than 10% of the vehicle treated control. A similar outcome was seen using S9 with L5178Y cells in this assay in the preliminary tests for selecting the range of concentrations performed prior to plating assessment.

Each photo is representative of 3 similar experiment with the same treatment concentration stained with Rapi-Diff staining. AAD double

Best Otc Drug Opiate Withdrawal

staining was carried

Best Otc Drug Opiate Withdrawal

out using SH-SY5Y and MCL-5 kratom concentrate wax cells treated with MSE and MIT as described in section 5. As translocation of phosphatidylserine to the outer plasma membrane indicates early apoptotic cell death Annexin V staining was used as a marker for apoptotic cells (van Engeland 1998). The cells become reactive with Annexin V prior to the loss of the ability of the plasma membrane to exclude 7-AAD staining and thus enables detection of unaffected (live) cells early apoptotic necrotic and late apoptotic cells (Darynkiewicz et al 2001). Each sample was analysed using Flow Jo 8.