I'm doing for cell surviving rate of breast cancer cell (MDA-MB-453) with Camptothecin (CPT)
I used RPMI 1640 for culture MDA-MB-453 (our lab did it for a long time)
and seeding to 96 well plate (10,000 cell/well) for 24hr,
After that, i use Camptothecin for several concentration (1nM~0.1mM) in 200uL and culuture 24 hr. (Camptothecin dissolved in DMSO 2 mg /ml store at -20 degree, and dilute in media before use)
Final, after i take out all of cell with trypsin 1x, and used trypan blue and cell (1:1) on Hemocytometer. I just can observe almost 70% survival rate even the highest concentration.
and I don't know why the different compare with previous research i read in paper. (Most research only 0.1nM~1uM)
Can anyone help me to solve the problem, thank you!
HI, Matloobi Zahra
Thanks for your input.
I checked the Camptothecin purchased from Sigma information sheet (dissolved in DMSO:10mg/ml).
but it is hard to dissolved in high concentration , so I used 2mg/ml in DMSO to make sure is dissolved completely.
and for example,if I want to used 60uM treat with cell , I taked 2ul of CPT and diluted in 200ul RPMI 1640 .
In addition , i think whether the pH value result in the high viability, but I just see a few of paper said about pH value influence in CPT treated with breast cancer cell. (Maybe they don't mention it.)
In my case, because the RPMI 1640 that I used stored for a long time will change the color to red means the pH value raised.
Is that a main factor to inactive my assay?
Thanks for everyone's reply.
yes PH value is important for breast cancer cell culture PH must be 6.4 - 7.4 in addition change in PH can change in CPT or any drug degradation and change your result .usually, culture medium stored 6 month and after this time it can be not appropriate , because characterization of its ingredients may be changed and reduce cell viability not only for PH changed
unfortunately I think you are mistake in adjust concentration properly
please explain me by detail
Hello Matloobi Zahra, thanks for your input.
Please correct my calculation if I'm wrong ,thanks.
First , I take 2mg of CPT dissolved in 1ml of DMSO as stock solution, so that concentration is about 5747uM.
than In the assay I use 96well to 0.001、0.1、1、10、100uM of CPT in 200ul/well ,respectively.
So i take 5.22 ul from stock solution of CPT add into 295ul of RPMI medium to get 100uM of CPT in medium,homogeneously.
Then I take 30ul of 100uM CPT in 270ul RPMI medium to get 10uM of CPT.
And 1、0.1、0.001uM of CPT used the same method to dilute and homogeneously.
348 gr/lit = 1mol
348 mg/ml = 1mol
348mg/ml = 1000mmol
2 mg/ml = 5.208mmol = 5208μmol
C1*V1 = C2*V2 5208x=300*100
30000/5208 = 5.76
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