I have a compound (C23N3OH27) to repeat some results with a molecular weight of 361.48. The problem is that the results are not being the same, I am evaluating cell viability (K562 and KG1) with resazurin (24 hours of plating 20.000 cells/100uL, 24 hours of treatment 100uL, 4 hours of resazurin 20uL) and the results lead us to believe that it does not induce death in any of the cases. concentrations tested (30 uM, 20uM, 10uM, 5uM, 1uM), I have already evaluated cellular metabolism, resazurin, interaction of the compound with resazurin and none explains the reason for not repeating the results. I am suspicious that it could be my dilution, I used a table from a colleague that performs the calculation automatically. Could someone help me to do the dilution directly just so I can assess if it's correct? I have 5g powder of the compound which was diluted in 2305.34uL of 100% DMSO, which according to the table gave me a solution of 6,000uM, I don't know if that's correct.
obs: my controls (+/-) are responding well so I don't believe it's the resazurin or the plating
Thanks for all contributions!
I have attached the dilution table below.
Hello Robson Araujo
Sorry! I did not understand the calculations from the excel sheet as it is very complicated.
“Could someone help me to do the dilution directly?”
Yes, let me make it simple.
The Molecular weight of the compound (C23N3OH27) is 361.48.
Then follow the sequence below.
361.48g -------- 1L -------- 1M
361.48g --------- 1L ------- 1000mM
0.36148g ---------- 1L ------ 1mM
361.48mg -------- 1000ml ------ 1mM
3.614 mg ----------- 10ml -------- 1mM
So, weigh 3.614mg of the compound in 10ml 100% DMSO to give 1mM stock.
You may prepare working solutions (30uM, 20uM, 10uM, 5uM, 1uM) as follows.
You may use the formula: C1V1=C2V2
C1= Concentration of stock solution (1mM)
V1= Volume of stock solution (X)
C2= Concentration of working solution (30uM)
V2= Volume of working solution (say 1ml)
Then,
1mM x X = 30uM x 1ml
1000uM x X = 30uM x 1ml
30/1000 = 0.03ml of stock i.e., add 30ul of stock solution to 970ul of media to give 1ml of 30uM working solution.
Similarly,
For 20uM
20/1000 = 0.02 ml of stock i.e., add 20ul of stock solution to 980ul of media to give 1ml of 20uM working solution.
For 10uM
10/1000= 0.01ml of stock i.e., add 10ul of stock solution to 990ul of media to give 1ml of 10uM working solution.
For 5uM
5/1000 = 0.005ml of stock i.e., add 5ul of stock solution to 995ul of media to give 1ml of 5uM working solution.
For 1uM
1/1000= 0.001ml of stock i.e., add 1ul of stock solution to 999ul of media to give 1ml of 1uM working solution.
Since 1ul is a very minute quantity to pipette, it may lead to error. So, you may dilute the stock by 1:10 to make a diluted stock (0.1mM). Then take 10ul of diluted stock (0.1mM) and add to 990ul of media to obtain 1uM working solution. Use this calculation for 1uM working solution instead of the above.
Best.
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