Biology Reference
In-Depth Information
22.2.2
MTT Assay
C6 glioma cells viability was determined by assaying the degree of MTT reduction.
1 × 10 4 cells/well was seeded in 96-well plate. Twenty-four hours later, cells were
treated with various concentrations of taurine (0, 1, 3, 9 mM) for 30 min prior to
treatment with 1 mM TOCP (dissolved in DMSO, the final concentration of DMSO
in the culture medium was 0.1% (v/v)). After exposure to TOCP for 48 h, 10 m l MTT
solution (5 mg/ml in phosphate buffered saline, PBS) was added into each well, and
the cells were further incubated for 3 h at 37°C. Afterwards, the media were aspirated
and 100 ml of dimethyl sulfoxide (DMSO) was added for the dissolution of formazan
crystals. The absorbance of each well was read at 570 nm using an ELISA plate
reader. Cell viability was expressed as a percentage of the control culture.
22.2.3
LDH Release Assay
Cells were treated as described above. After various treatments, the amount of LDH
released into the medium was determined using a diagnostic kit (Nanjing Jiancheng
Bioengineering Institute, China). In brief, NADH and pyruvate (0.1%, w/v) were
added and the samples were incubated at 37°C for 15 min. Next, the samples were
incubated with the coloring reagent for 15 min. The reaction was stopped by adding
0.4 mol/l NaOH, and the activity (U/ml) of LDH in each sample were calculated from
formula. The LDH release was expressed as a percentage of the control culture.
22.2.4
Morphological Observation
To examine the cellular morphology of C6 glioma cells, cells were seeded into
24-well plates. After various treatments as described above, cells were observed by
phase-contrast microscope.
22.2.5
Measurement of GSH Content and GPx Activities
The content of GSH and the activities of GPx were all determined by using assay
kits (Nanjing Jiancheng Bioengineering Institute, China). GSH was measured based
on that 5,50-dithiobis (2-nitrobenzoic acid) (DTNB) reacts with GSH to generate
2-nitro-5-thiobenzoic acid and GSSG. The concentration of GSH was calculated
from formula and expressed as nmol per milligram protein. The assay for GPx
activity was assayed by quantifying the rate of oxidation of the reduced glutathione
to the oxidized glutathione by H 2 O 2 catalyzed by GPx. One unit of GPx was defined
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