Material Methods Essay Example

- Cell Culture and Cell Lines:
Three breast cancer cell lines: CAL120, EVSAT and MDA-MB-231, were cultured individually, in RRMI 1640 supplemented with 20% Fetal Bovine Serum (FBS), 100U/ml Penicillin, 100U/ml Streptomycin and L-Glutamine. The cells were cultured and maintained in an incubator at 370C supplied with 5% CO2. For subsequent experiments, the cells were further cultured till confluence, in 6 well and 96 well plates using RPMI+20%FBS.
1.2 Treatment of Cells:
For treatment, 1x106 cells were seeded in 6 well culture plates, and incubated for 24 hrs. After 24 hours, the old media was replaced with a fresh media containing 5% FBS. Cells from 3 well each were used for the control, not treated and treated with varying doses of Embelin. Following treatment, the cells were detached by trypsinized and collected for further viability, apoptosis or protein analysis.
1.3 (4, 5-Dimethylthiazol-2-yl)-2, 5-Diphenyltetrazolium Bromide (MTT) Assays:
For MTT assay, 1x104 cells were seeded in 96 well plates. Six wells each were used for control, treated and not treated. The cells were treated with 200µM of Embelin, ranging from 5nM up to 200nM for 24 hours. After the Embelin treatment, the cells were incubated with MTT dye (55µg/ml) for 4 hours. At the end of the incubation, cells were collected by centrifugation at 1000RPM for 10min. The cell pellets was resuspended in 200ul of 100% isoproponol and incubated at room temperature (RT), overnight with gentle agitation. The cell viability was then measured using electronic plate reader.
1.4 Annexin V staining and Apoptosis detection:
The breast cancer cell lines were treated with Embelin as mentioned above in 1.3. After treatment the cells were incubated in Annexin V and Propidium Iodide (PI) for 30 minutes at RT. The apoptosis was then analyzed by flow cytometry. The cells staining red are positive for PI and are necrotic, while apoptotic cells are positive for Annexin V and stain green.
1.5 Cell lysis and protein preparation:
The breast cancer cell lines were treated with Embelin as mentioned above in 1.3. After treatment, the cells were trypsinized and pelleted by centrifugation. The pelleted cells were then washed once, by re-suspending in 1X Phosphate Buffered Saline (PBS). The washed cells were again pelleted by centrifugation at 1000 RPM for 10 min at 40C. The cell pellets were then re-suspended in phosphorylation lysis buffer (0.5-1.0 % TritonX-100, 150mM NaCl, 1mM EDTA, 200M Na3VO4, 10mM Na2H2P2O7, 100mM NaF, 1.5mM MgCl2, 1mmol/L PMSF,10g/ml Aprotonin) and incubated on ice for 1 hour. The suspension was then centrifuged at 14000RPM for 15 minutes at 40C. The supernatant was collected and the protein concentration was estimated.
- Western Blotting:
For western blotting the proteins were separated in 10% SDS-PAGE for 2-3 hrs, at 90V. Molecular weight marker was loaded in the first well and equal concentrations of proteins were loaded in the other wells. The proteins were then blotted on to a polyvinylidenedifluoride (PVDF) membrane (Immobilion, Millipore) for 1 hour at 40C.The PVDF blot was then blocked using 5% skim milk powder for 1 hour and then incubated overnight at 40C with the primary antibody. The blot was then washed 3 times in 1x TBST, 15 minutes each and then incubated with secondary antibody for 2 hours. The final wash was done in TBST as mentioned earlier. The blot was then probed using a chemiluminescence kit to detect the protein of interest.