Hi All,
I've been treating several cancer cell lines and a non-cancer cell line with WST-1, a variation of the MTT or MST viability assays, to monitor cell death with my immunotherapy drug. All cell lines I'm using convert the reagent as expected, except for the MCF7. Under the microscope, my MCF7 cells are visibly dead after treatment, but the absorbance readout in the treated conditions goes up, not down.
Can anyone recommend an alternate assay to assess viability of MCF7 cells that does not use the same substrates as this line of viability assay?
Thanks!
Allyson
The reason the absorbance goes up, is the dye is still leaking out of your lysed cells, even after death. MTT assays are a bit noisy. The dye may be attaching to your T cells too? If you want to stick with an endpoint assay, you may want to try the LDH assay, which uses formazan dye. Will this work? Promega has a kit. Or if you want check out our impedance system for live cell, dye/kit free immunotherapy cell death analysis, in real time - you just need to convince your PI! Good luck! Debby
Dear Allyson
I have used a clonogenic assay or colony formation assay with good results.
It was first developed by T.T. Puck and Philip Marcus at Univ. of Colorado in 1955.
It will allow you to better see cellular senescence as well observe the cytotoxicity.
Which can be difficult to understand from standard MTT assays.
They are cheap, and effective. They can be a little more labor intensive compared to using a plate reader to count vs a stereoscope and your eyes.
I attached one of my old protocols in case you have any interest for your project.
Best Wishes!
You could use alternatively SRB assay or Cristal Violet assay to evaluate cell viability.
SRB assay:
Article Sulforhodamine B colorimetric assay for cytoxicity screening
Cristal Violet Assay:
Article Crystal Violet Assay for Determining Viability of Cultured Cells
Thanks everyone!
Charles D Anderson does the assay you mentioned target a specific senescence marker in the cells? And how is it measured?
Each method evaluates different aspect of cellular responses.
Colony formation assay analyzes the cellular clonogenic potential.
As the MTT and MTS will give a window to the cellular metabolizm.
No specific marker, but visual counting of all cells alive, and able to make a colony.
A failed colony is one that is only a few cells, and is easily seen when stained compared to a colonies that is not.
A or example, prematurely senescent cells are clonogenically inactive (do not form colonies), but are metabolically active and viable.
Most recent answer. MTS, MTT or similar assays of this sort evaluate metabolism, whereas colony formation assay evaluates clonogenicity. ... Metabolic state and clonogenic potential are not necessarily the parallel events, such that prematurely senescent cells do not form colonies but are metabolically active.
Oh, sorry...
It is measured by visual counting of colonies from cells.
Seed at low density, treat with exp. Rx...
then count and compare to controls or no Rx.
The less the number of cells of Rx to Control
the more cytotoxic your Rx.
standard colony formation assay, its been used for a lot of
cancer cell lines and compounds.
Thus there is a lot of data to compare
yours to, as to get a good starting point of things like
seeding density, and Rx concentrations to start with.
UPDATE - thank you to everyone for their suggestions. I ended up modifying a crystal violet staining protocol to use in a 96-well plate and plate reader. It works really well for all of my adherent cell lines. If anyone would like to see the protocol, I would be happy to share it!
Hello, Dear Allyson Cochran can I see your protocol of crystal violet used to read plate (96-well plate)
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