Hello everyone,
I've tried a few XTT protocols from our lab,but I've been getting negative results from reading the 96-well plate when trying to measure splenocyte proliferation.
I follow this simple protocol after loading approximately 2X10*5 cells per well-
Add a volume of reconstituted XTT Stock Solution
equal to 20% of the culture medium volume to be
tested.
Return cultures to incubator for 2–4 hours
depending on cell type and maximum cell density.
An incubation period of 2 hours is generally
adequate, but may be lengthened for low cell
densities or cells with lower metabolic activity.
Incubation times for similar samples should be
consistent to make comparisons.
Measure the absorbance at 450 nm. Tests
performed in multiwell plates can be read with an
appropriate plate reader. Alternatively, the contents
of an individual well may also be transferred to an
appropriate cuvette for spectrophotometric
measurement.
Note: When multiwell plates are read with an
appropriate plate reader, it is recommended to also
measure the absorbance at 690 nm. Use the
absorbance at 690 nm as a background
measurement and subtract it from the 450 nm
value.
I know that because these cells do not adhere to the well,I'm obliged to use the centrifuge and collect the supernatant in order to read the results,but unfortunately reading from the new plate shows no difference from control.
Is anyone familiar with this setback? or knows a good XTT protocol for splenocytes which I can try?
Thank you!
Hi Ido
I didn't understand why you are reading on a new plate. When I ran MTT assays I just centrifuged my plate, carefully removed the media and solubilized the crystals with DMSO (on the same plate). As far as I know you don't need to remove the cells on XTT assay.
Take a look at this ATCC protocol. It also has some troubleshooting.
https://www.atcc.org/~/media/56374CEEC36C47159D2040410828B969.ashx
One my college tried to use MTT for same reasons with same results. And I have to say it is not very good way to estimate proliferation of mixed population with huge amount of dead cells.
Depending of your protocol you will get information mainly about metabolic activity of cell population OR reactive oxygen species (ROS) production + metabolic activity depending of using OR not of electron carriers together with XTT. Phagocytes during ROS production could account to growth of XTT signal much more than metabolic activity byself if no electron carriers was added (please read attached file).
If you still intended to use XTT for estimation of cell proliferation at first be sure what all cells in the very start of cultivation was alive. Test viability of cells also in the very end of cultivation. It will gives you some ideas about how many cells proliferate, how many died and when it could happens.
May be after all you will try to find other way to estimate proliferation, f.e. flow cytometry or labeled incorporation.
Have a nice experimental work!
Conference Paper THE PECULIARITIES OF EVALUATION OF OXIDATIVE METABOLISM IN C...
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