EZ4U - Cell Proliferation Assay

Protocol tips

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	Add 200 µl cell culture into respective wells. Add 20 µl SUB (substrate) into each well, swirl gently. Incubate at 37°C for 2-5 hours,

Protocol tips
Add 200 µl cell culture into respective wells. Add 20 µl SUB (substrate) into each well, swirl gently. Incubate at 37°C for 2-5 hours,

Publication protocol

The cells were subjected to the trypsinization process and then suspended at 4 × 105/mL in complete F-10 medium. 50 μL aliquots of cell suspension (2 × 104 cells) were placed in the wells of plastic 96-well culture plates (96 Cell Culture Cluster Dish, Costar; Nunclon, Microwell Plates, NUNC). After 12 hr period of preincubation (5% CO2, 37°C, 95% humidity), the tested substances were added to the appropriate wells: in experiment (1), ang II or ang IV at final concentrations of 10−6 M, 10−8 M, 10−10 M, or 10−12 M; 10−8 M ang II or 10−8 M ang IV + amastatin at final concentrations of 10−5 M, 10−6 M, or 10−7 M. The appropriate volume of the culture medium was added to the wells of control group and to the wells with one tested substance to the final volume 200 μL in each well. In experiment (2), ang II at final concentration of 10−8 M, ang 5–8 at final concentration of 10−10 M, PD98059 at final concentration of 10−5 M, SB203580 at final concentration of 10−5 M; 10−8 M ang II or 10−10 M ang 5–8 + 10−5 M PD98059; 10−8 M ang II or 10−10 M ang 5–8 + 10−5 M SB203580. Both PD98059 and SB203580 were dissolved in the DMSO/medium solution, in the proportion DMSO : medium 37 : 63 for PD98059 and 1 : 9 for SB98059. Since DMSO is known to affect cellular viability, the appropriate solution of culture medium with DMSO was added to the wells of control group and to the wells with ang II or ang 5–8 alone, to obtain the final DMSO concentration equivalent to that in the PD98059- or SB203580-treated groups. Finally, the appropriate volume of the culture medium was added to the wells of control group and to the wells with one tested substance, to the final volume 200 μL in each well.

After 72 hrs of incubation (5% CO2, 37°C, 95% humidity), cell viability was estimated using the modified Mosmann method, following the procedure recommended by the producer of the kit (EZ4Y, Easy for You, the 4th Generation Non Radioactive Cell Proliferation and Cytotoxicity Assay, Biomedica Gruppe, Austria, Bellco Biomedica Poland). The optical density (OD) of each sample was measured by a microplate reader at 450 nm.

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