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I am using Greiner Thincert Culture Inserts for 6-well plates and A549 cells in my exposure experiments. The goal is to expose the cells at an air/liquid interphase for a certain period and to measure cell viability, oxidative stress markers and cell cycle stage after the exposure.
Until now I have tried to detach the cells from the inserts and measure the MTT assay and do flow cytometry analysis. However I have been having problems detaching the cells from the inserts, trypsin/EDTA is ineffective and scraping destroys the cells, so I have not been able to get sensible data from our exposure experiments.
Can you recommend any kits/procedures which could be applied directly in the inserts without prior detachments of the cells?
The minimum parameters I would need to evaluate after the exposure are cell viability and apoptosis/cell cycle stage, but I would like to include a marker for oxidative stress (e.g. glutathione) as well. I should be able to measure all the mentioned parameters simultaneously in one insert without too much quenching and fluorescence interference/overlay.
We have a Victor III microplate reader and an inverted fluorescence microscope for which we will acquire quantification software shortly.
Thank you for your help.
With best regards,
Asked on Mar 06 2012
Thank you for your enquiry and interest in our products.
MTT assay can be carried out on 96 well plates using either adherent or floating cells. You do not necessary need to remove (harvest) the cells by trypsinization. You can culture the cells in the wells of the 96 well plates, then apply the MTT solution (20ul) to each well. During incubation (37C, 5% CO2) for 1-5 hours MTT will be metabolized. The formazan (MTT metabolic product) can then be dissolved in DMSO and the optical density can be read at 560 nm using a plate reader - without harvesting the cells. There may be other cell viability/cytotoxicyprotovols but this is the most widely used.
Cell viability kits:
We have different cell viability assay kits in our catalogue i.e. ab112120, ab112121, ab112122, ab112123.
GSH can be detected/quantified either by fluorimetric or chromogenic (DTNB - Ellman's reagent) assays and currently we have also some assay kits for detecting this tripeptide, you may wish to take a look at the datasheets for further information.
- ab65322: Glutathione Fluorometric Detection Kit
- ab112132:Intracellular GSH Assay Kit
Normally, intracellular GSH levels can be expressed per cell numbers or per protein concentration.
I hope this helps and if I can assist further, please do not hesitate to contact me.
Answered on Mar 06 2012