Question

-The probe stock was provided in DMSO and water. DMSO is toxic to cells at a high enough concentration. Calculate the final concentration of DMSO in your dosing solution, and look up what concentration is considered the threshold to use in cell culture assays (cite your reference).

Some helping information from my pre-lab:

According to the information, 16 wells of the plate are required to be filled with a solution containing a requisite percentage of methanol along with culture media. In order to make the required concentrations of solutions, following calculations can be made:

0% Methanol: Add 16*100ul i.e. 1600ul or 1.6ml of culture media.

1% Methanol: Add 1600*1/100 i.e. 16 ul of methanol to 1600-16= 1584 ul of culture media.

5% Methanol: Add 1600*5/100 i.e. 80 ul of methanol to 1520 ul of culture media.

10% Methanol: Add 1600*10/100 i.e. 160 ul of methanol to 1440 ul of culture media.

20% Methanol: Add 1600*20/100 i.e. 320 ul of methanol to 1280 ul of culture media.

40% Methanol: Add 1600*40/100 i.e. 640 ul of methanol to 960 ul of culture media.

70% Methanol: Add 1600*70/100 i.e. 1120 ul of methanol to 480 ul of culture media.

These calculation will made the requisite volumes

. The ethidium homodimer-1 (EthD-1) probe is supplied by the manufacturer in a concentrated stock solution of 2 mM in DMSO/H2O 1:4 (v/v). The final working concentration of probe in the dosing solution should be 2 µM. What is the corresponding dilution factor, and specifically how much of the probe stock has to be added to the dosing solutions from question 3? ​=1.6 micro liters

Answer 1

DMSO is an organosulfur compound which is used in cell banking applications as a cryoprotectant. High concentrations should never be used for cell culture. 5% is very high and will be dissolving the cell membranes. Most cell lines can tolerate 0.5% DMSO and some cells can tolerate up to 1% without severe cytotoxicity.

Rule of thumb, 0.1% DMSO is considered to be safe for almost all cells. 0.5% DMSO as the final concentration has been used widely for cell culture without cytotoxicity.

The stock concentration of probe is 2mM in DMSO (v/v). The final concentration of probe is 2uM in volume of 1.6ml cell culture media.

M1 x V1 = M2 x V2

2mM x ? = 0.002mM x 1.6ml

V1 = 0.002 x 1.6 / 2 = 0.0016 ml or 1.6uL

1.6uL of probe which is dissolved in 100% DMSO is being diluted in cell culture media to 1.6mL. The dilution factor is 1000, which gives a final concentration of 0.1% in each well.