Cytotoxicity assay

Using cell culture medium as the diluent, prepare H₂O₂ solutions with concentrations mM. For the experiment we will need at least 1.5 mL of each solution.

Material:

Procedure:

Each solution must be prepared by dilution of the previous one, so that its concentration is the desired one and there will be enough to prepare the next as well as to use in the experiment three 500 µL aliquots each (and some excess to spare).

Part 2: MTT assay (metabolic viability)

Fundamentals

Material:

24-well plate, with PC3 cells adhered to the bottom of wells.
Micropipettes and sterile tips.
H₂O₂ solutions prepared previously.
0.5 g/L MTT solution: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, in RMPI medium.
0.2% SDS (sodium dodecylsulphate).
Dimethylsulfoxide (DMSO)
Cell culture medium, warmed to 37°C: RPMI medium supplemented with 10% foetal bovine serum and 1% antibiotics.

Treatments:

Negative cytotoxicity control (⊖): without H₂O₂
Positive cytotoxicity control (⊕): with 0.2% SDS
Test: with diverse H₂O₂ concentrations
Everything is assayed in triplicates.
show a possible arrangement of the treatments in the plate. Write down in your lab notebook this or any other arrangement you decide.

Instructions to use the virtual laboratory:

Click on the source tube, from which you want to take liquid, and the micropipette will move to it and will aspirate. (If you make a mistake, click on the waste bin to discard the tip with its contents.) To deliver the content of the micropipette tip, click on the target well in the plate, where you wish to add the liquid.

Procedure:

To start, culture medium from the wells (it is enough to turn the plate upside down and shake to drain the liquid).
Add to each well 500 µL of one of the treatment solutions: medium (⊖), SDS (⊕) or H₂O₂, according to the planned arrangement.
for 30 minutes inside the cell culture oven (5% CO₂, 95% air, 90% humidity, 37°C).
the content of the wells.
Add to each one 500 µL of the MTT solution.
for 2 h to allow for the reduction of MTT.
agan the content of the wells.
Add to each one 500 µL of DMSO, which will dissolve the newly formed, purple, formazan.
absorbance at 570 nm in the plate reader.
Copy results and prepare with them a graph where you plot %viability versus H₂O₂ concentration of the treatment (use logarithmic scale for the concentration).