I am working with LNCaP cell line. I have done several trials of MTT assay to test the cell viability with different concentrations of docetaxel at the range of uM to nM. Unfortunately, there were no to little toxicity to the cell line. I used different numbers of cell: 2000, 4000, 5000, E4, and 5E4 cells/well, but there was no difference. I also seeded the cells for 24 or 48 hours before docetaxel treatment, and again, no toxicity.
I also used needle to passage the cells in order to break the clumps, which was based on other people's suggestions on another person's profile. This helped when I seeded the cells on the flask, but it was not helpful for seeding cells on 96-well plate. The cells did not form monolayer in 96-well plate. I am not sure what I can modify the procedure to determine the cell viability test.
Please if any of you can provide any suggestions or recommendations, I truly appreciate it very much.
Thank you.
Hi
ٰYou can use 10000 cell in each well and in MTT test after adding dmso wait for 2 hours then use ELISA reader for reading results
Hi Matloobi,
Thank you for your suggestion. I tried to use 10,000 cells/well, but I still could not get data for cell viability test. Based on the literature, the IC50 of docetaxel in LNCaP is around 1.6 nM. I do not understand why there was no toxicity of docetaxel to LNCaP cell for my experiment. Could you give me some advises?? Thank you.
Have you verified that your docetaxel is active on other cell lines? Some preparations of docetaxel may be very difficult to get into solution. Are you starting with a powder or a ready to use solution of docetaxel? If you prepare a suspension rather than a solution and then sterile filter it, you will end up losing most of the docetaxel. I would first verify that you docetaxel is fully in solution and active before proceeding.
Hi Alan,
Thank you for your response. Yes, I tried use docetaxel in PC3 cell line, and it works fine. The drug was dissolved in DMSO as 1 mM stock, aliquoted and stored at -20C. Before the experiment, it was diluted into 1X PBS to 100 uM as a working stock. I usually used the aliquot right away and stored the unused one at room temperature. I will check the solubility again for sure. Thank you!!
Hello,
I am also doing the cell viability test of docetaxel in LNCaP. What do you mean about no toxicity? My problem with MTT assay in LNCaP is mainly caused by the clumping of cells. I tried in 96-well plate and 24-well plate, the results variation is big. But I can calculate the IC50 of Doc which is around 5 to 10 nM, when LNCaP culture for 5000 cells/well, 72h.
Does the LNCaP cells you cultured grow well? In a healthy and monolayer status?
And by the way, you mentioned the IC50 of docetaxel in LNCaP is around 1.6 nM. Could you please show me where do you find this data?
Thank you!
Hi there,
Yes, I also have problem with the clumping cells.But, I used needle to passage the cell into the medium before I seeded them on the 96-well plate. The cell was health and in the monolayer form. However, the results that I got were consistent with 10-25% of the toxicity throughout the DOC concentration range from pM to uM. I tried with 5000 or E4 cells/well for 72 hrs and the results were similar.
Can you please let me know how you did your MTT assay?
The reference was from Mol. Cancer Ther, 2011, 10(9), 1728-1739.
DOI: 10.1158/1535-7163.MCT-11-0191
Thank you!!
Hi HSUAN,
I hope you find the answer for your trouble by this time. To share info-
Trypsinization of LNCaP cells is tricky which can cause clump formation.
If we are constantly maintaining them in Log phase-
1) it is easy to detach them by using very less tripsin (100ul commercial trypsin-EDTA+900ul of cold DPBS)(more trypsin and its incubation time=clump formation) ;
2) lesser speed for pelleting (~1000/2min);
3) distribute cells into plate after resuspending cells in the media (12 well =1ml to each)
4) try not to disturb plate after seeding at least for 12hr.
With healthy and monolayer LNCaP/ LNCaP stable cells we get Docetaxel IC50 1.5- 6.0 nM.
- Badges
- Science topic