All of the reac tions were performed in 50 uL reaction volumes in trip licate. Standard curves were generated for gC1qR and B actin. The B actin gene was used as an internal selleck chemicals llc control in all of the PCR e periments. Western blot analysis After various treatments, cells were harvested, pelleted by short centrifugation and suspended in lysis buffer sup plemented with protease inhibitors for 30 min on ice. The supernatants were collected by centrifugation at 13,000 g at 4 C for 15 min. An equal amount of pro tein was separated by SDS PAGE on a 10 15% polyacryl amide gel and transferred to a PVDF membrane. The transferred membranes were blocked for 1 h in 5% non fat milk in PBST, in cubated with appropriate primary antibodies followed by horseradish pero idase conjugated secondary antibodies.
The protein bands were visualised using the enhanced chemiluminescence Western Detection System. Cell viability analysis The water soluble tetrazolium salt assay was performed to as sess C33a and SiHa cell viability. The WST 1 assay is a colorimetric method in which the dye intensity is propor tional to the number of viable cells. Cells were seeded into 96 well microtitre plates at a concentration of 5 103 cells well. After 12 h of incubation, cells were treated with for 48 h. After incubation, the cells were washed with PBS, WST 1 cell proliferation reagent was added, and the sam ples were incubated for 4 h. Sample absorbance was analysed with a bichromatic ELISA reader at 450 nm. All of the e periments were performed in triplicate with dif ferent C33a and SiHa cell passages.
Cell migration analysis C33a and SiHa cell migration was measured using 24 mm diameter chambers with 8 um pore filters. Cells were collected and resuspended in serum free media, and a 0. 2 mL cell suspension was added to the upper cham bers. Treatment media was added to the lower chambers. The chambers were incubated for 48 h at 37 C in a humidified atmosphere of 5% CO2 95% air. Ne t, the filters were fi ed in 95% ethanol and stained with H E. The upper filter surfaces were scraped twice with cotton swabs to remove non migrated cells. E peri ments were repeated in triplicate with different passages of the C33a and SiHa cells, and the migrated cells were counted microscopically in five different fields per filter. Apoptotic cell detection C33a and SiHa cell apoptosis was detected using the Anne in V FITC propidium iodide staining kit via flow cytometry.
After different treatments at the indi cated times, C33a AV-951 and SiHa cell were washed and resuspended in binding buffer before being trans ferred to a 5 mL tube. The cells were incubated in the dark with 5 uL each of Anne in V FITC and propidium iodide for 15 min. Binding buffer was then added to each tube, and the samples were analysed using a Beckman Coulter Epics L flow cytometer.