Dear Researcher,
currently I am searching for an alternative Assay to detect cytoxicity of drugs against the cell line MDA-MB-231.
1) So far, we used CellTiterGlo (Luminescence signal for ATP) in a 384- well format, but in the 96-well format we would have to largely reduce the cell density (which would be below 1x10e5 cells/well). Total duration is: 48 h (24h seeding + 24h compound treatment).
--> Are there any experiences concerning this Assay that could help me?
2) Can you recommend any cytotoxicity Assay which worked out quite well in a 96-well format using this cell-line? Are there any recommended cell number?
3) Further, are there any suggestions for a positive control (and concentration) In this or another cytotoxicity Assay?
I'd be pleased about every suggestions from you! Thanks a lot in advance!
Hi Irene Reimche
For 96-well format and this cell line I suggest XTT or MTT test for the cellular toxicity assessment.
Hi Irene...have you tried to test the Sulphorhodamin B assay it is a good protocol to evaluate cytotoxicity when classic MTT protocols fail or got dubious results...
Muhammad Haddad Carlos Frederico Leite Fontes Thank you for both suggestions! So far I only tried out CellTiterGlo luminescence Assay.
Hi Irene,
I can recommend the AlamarBlue-Assay. I incubate cells after treatment for 2h with the AlamarBlue-solution and analyze fluorescence per photometer. If it is necessary it is possible to cultivate the cells for further period because it is non-toxic. For positive control I kill the cells with 60% isopropanol (few minutes) or phosphoric acid during the whole treatment (24h).
You could use alternatively SRB, Crystal Violet, Neutral Red (Absorvance) or Alamar Blue (Fluorescence) assays to evaluate cell cytoxicity.
Crystal Violet is the easy and quick as you can see in this protocol.
Hi Irene Reimche,
The LDH or MTT assay is suitable.
To gain more experience, it is better to use several of cytotoxicity assay and compare the results with each other.
You can use doxorubicin for positive control.
You can also measure the effect of your combination on a normal cell line. For example, MCF10A cell line.
The MCF10A is a non-tumorigenic epithelial cell line.
I think that LDH measurement is a good way to measure drug cytotoxicity as well as trypan blue exclusion. MTT assay is not really in my opinion a cytotoxicity test but a determination of mitochondrial activity.
Hello Irene,
the MDA-MB-231 cell line has been used for the assessment of cytotoxicity by Theodossiou et al. in 96-well format in the following publication: "The Diverse Roles of Glutathione-Associated Cell Resistance Against Hypericin Photodynamic Therapy", Redox Biol, 12, 2017,191-197 (doi: 10.1016/j.redox.2017.02.018).
Hope that helps - CL
Thank you all for the precious answers! Intersting, that trypan blue and crystal violet staining is used for a cell viability assay. The Resauzrin based assays seem beneficial in regard to measurement during the whole experiment.
I'm worried for my positive control, because some chemotherapeutics don't work or at least work in a high concentration. Does someone has experiences concerning the concentrations?
You could do a dose-response experiment with the chemotherapeutics and your cells to know which concentration you can use. Because the concentrations that are cytotoxic to your cells can be different from the ones from another lab , just because they use a different cell culture media or a different FBS concentration to maintain the cells,etc...
For example for MDA-MB-231, a positive control with Doxorrubicin 5 uM will get you a decrease in cell viabity around 70-80%. Article Anti-Migratory and Pro-Apoptotic Properties of Parvifloron D...
But as I said you may need more or less to have the same effect on your cells.
WST-1 cells proliferation kit assay works perfectly with MDA-MB 231 breast cancer cells
Hi Irene: We just published a relevant article comparing several cytotoxicity assays, including the strengths and weaknesses of the assays. We also describe a specific assay (the RNA disruption assay) that quantifies ribosomal RNA degradation upon cell death. This assays appears well-suited to quantify cytotoxicity but not cell cycle arrest. Our paper in Scientific Reports is now available online.
https://www.nature.com/articles/s41598-020-65579-9?utm_source=other&utm_medium=other&utm_content=null&utm_campaign=JRCN_2_LW01_CN_SCIREP_article_paid_XMOL
Kind regards,
Amadeo Parissenti
I agree with Dominique Duval that MTT is not really a cytotoxicity assay. In the late 80's, I had the pleasure to meet Tim Mossman who developed the MTT assay and inquire with him about it. He developed it for monitoring hybridomas - not as an assay to monitor the effect of compounds on cells. In my experience at a previous company, I had test compounds that would boost the MTT signal but were antiproliferative by 3H-Thymidine incorporation. So the test compounds were having a direct effect on the mitochondrial activity in the cell population originally plated.
Keep in mind that concentrations of drug required to arrest the growth of cells (or to affect plasma membrane integrity/mitochondrial respiration) are often much lower than concentrations required to induce cell death (as measured by DNA content analysis or our RNA disruption assay).
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Cell Biology
- Culture Cells
- Cell Line