Hello Heemin Lee

For H2O2 treatment:

Instead of 3%H2O2, you may use 30% H2O2 for the treatment of cells. 30% H2O2 means that for every 100ml of the solution, there is 30g of hydrogen peroxide. To prepare 1M of H2O2, you need the molar weight in 1L. So, for 1M H2O2, you will need to dissolve 34.01g of H2O2 in 1L. Since H2O2 is a solution form, 30g is present in 100ml of water. By calculation, 34.01g will be present in 113.37ml.

So, you may take 113.37ml of the 30% solution and make up to 1L with water to make a 1M solution of H2O2. You may reduce the volume to 10ml instead of 1L. So, you may take 1.13ml of the 30% solution and make up to 10ml with water. Treat this 1M solution of H2O2 as the stock solution.

You require 200uM as the working concentration. So, you make 1000uM H2O2 as the diluted stock by diluting the stock(1M) 1000 times with media.

For 6-well plate, the volume required per well is 2ml. So, you will take 0.4ml of diluted stock (1mM) and add 1.6ml media, mix and add to the cells in the well to give 200uM H2O2 in 2ml.

For CuSO4 treatment:

To prepare 1M of CuSO4, you need the molar weight in 1L i.e., for 1M CuSO4, you will need to dissolve 249.69g of CuSO4 in 1L. You make a stock of 1M (1000mM) in 10ml by weighing 2.49g of CuSO4 in 10ml media.

Make a diluted stock of 1mM by diluting the stock 1000 times with media.

For 6-well plate, the volume required per well is 2ml. So, you will take 1.5ml of diluted stock (1000uM) and add 0.5ml media, mix and add to the cells in the well to give 750uM copper sulphate in 2ml.

Best.

Hydrogen Peroxide (H₂O₂) Treatment

Preparation:

• Stock concentration: Most commercial H₂O₂ solutions are 30% (w/w), which is ~9.8 M. Check your product details.
• Dilution: It’s recommended to prepare a fresh working solution in sterile PBS or culture media before each experiment.

Steps for Dilution: