Dear all,
I am trying to determine the IC50 of various phytonutrients in a panel of breast cancer cell lines. I treat cells with various phytonutrient concentrations. Then try to determine cell viability at the start of the treatment (t0) and after 72 hours.
I use home-made resazurin (62.5 uM) for this assay. I add 4.8 ul of my stock resazurin to 100 ul of medium/cells directly. After 2 hours incubation at 37C, I read the fluorescence. Even though I could see that there were dead cells at higher phytonutrient concentration treatments, the fluorescence value was very similar to the vehicle treated control.
Can someone help me figure out what may be going on here?
Thank you
NTP
Hi.
I work with lymphocyte. To determinate limphocyte proliferation in culture I use 25uL of risazurin (o,01%) to 200uL of medium/cell directly after 48h. After 24h incubation I read color and I get great diference. (600 e 540 nm)
I suggest more hour of incubation, because resazurin reduces to resofurin in culture conditions and than we can see color or fluorecence.
We found that the cells that you use have to have a high metabolic activity for this assay to work. In the original papers, the cells were actually stimulated and then treated. We found that primary cells, neuronal stem cells, in culture did not give any change in signal with resazurin, even if we left it on for 24 hrs. As Brenda Parsons said, we had to switch assays. The MTT assay also requires the cells to have a relatively high metabolic activity. We chose a crystal violet assasy, which has several more steps, but it does not depend on mitochondrial or enzyme action within the cells.
Good luck
Hello All,
I have decided to test sensitivity as suggested by Brendon, and run a timecourse to determine when is the best time to get a good readout.
I would be interested in getting the Crystal violet protocol from Sarah.
Thank you so very much
Nupur
Crystal Violet (color is predominantly in cell nuclei)
Fix cultured cells with 4% glutaraldehyde in phosphate buffered saline (PBS) (pH7.2): 10 mins room temp., rinse 3 times with PBS (or water), remove. Add 1% crystal violet (Fisher C-581) in water. (100 ul/96 well, 0.5 ml/12 well) Incubate 30 mins, rm temp. Remove and rinse gently AT LEAST 3 times with water (important to rinse (gently) until color stops coming out). Air dry. Add 10% acetic acid in water. (same volumes). Gently agitate for 10-30 minutes. Read on plate reader at 590 Abs.
Methods, references for crystal violet and example of cells that don't give a signal with resazurin are given in: Reference: Vennemeyer, J., Hopkins, T., Kuhlmann, J., Heineman, W.R., and Pixley, S.K. (2014) Effects of Elevated Magnesium and Substrate on Neuronal Numbers and Neurite Outgrowth of Neural Stem/Progenitor Cells in Vitro. Neuroscience Research 84:72-8.
Good luck!
Sarah
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