Dear all,
I would like to ask, if DMSO can be a positive control in this particular experience.
L929 murine cells stimulated by murine-TNF-Alpha by 8 hours mainly leads to cell death by Necroptosis. And using Necrostatin-1 (Nec-1) I can completely revert the enzymatic signal increase do to complete protection of membranne leakage. Here I'm using Lonza Toxilight for measuring Adenilato Kinase (AK) realease to medium.
And My question regards to the luminencense value given by the Glomax reader.
Assay conditions and respective AK RAW values:
Na - 3000
DMSO - 3000
TNF-a - 9000
TNF-a + Nec-1 - 2000
if I turn it into fold to DMSO (as positive control) to evaluate Cell membrane leakage/permeabilization:
NA - 1
DMSO - 1
TNF-a - 3
TNF-a + Nec-1 - 0,6667
if I turn it into fold to Nec-1 (as positive control) to evaluate Cell membrane leakage/permeabilization:
NA - 1,5
DMSO - 1,5
TNF-a - 4,5
TNF-a + Nec-1 - 1
My question is that in this paticular case Nec-1 values are under DMSO or NA, and biological meaning of this is dificult to understand.
And If I run a screening to identify new necroptosis inhibitors, in theory rational says that a positive control (produce the same result in the assay as a desired active compound). Here using AK realease as cell death assay, and given the fact that Nec-1 give me a value under the DMSO or NA. Is it correct to use the DMSO as positive control and DMSO + TNF-a as negative control to calculate the % of inhibition of new tested compounds using this formula for RAW values:
100 x (((TNF-a) - (TNF-a + Nec-1/Tested Cmp)) / ((TNF-a) - (DMSO)))
Thanks for your Help
Cheers
One thing to consider is that DMSO quenches fluorescence. See, for example (among others)
Agents Actions. 1988 Jun;24(1-2):35-9.
Dimethyl sulfoxide decreases the fluorescence yield of the reaction between histamine and ortho-phthalaldehyde and may influence histamine release.
Moldt P1, Andersen WK, Christensen SB
From what I understand, you are trying to identify new necroptosis inhibitors. What is NA? If you are trying to identify a necroptosis inhibitor, your negative control would be cells treated with TNF and your positive control would be cells treated with TNF + Nec-1. You then normalize your data to the untreated cells.
I understand that your untreated cells (NA?) or DMSO treated cells (baseline controls) have OD values lower than the cells that were treated with TNF + Nec-1. We have observed this before and it could be because of low levels of cell death/necroptosis happening in cells in the absence of stimulation, which Nec-1 inhibits. See the paper linked below and you would notice that the viability for zVAD + Nec is higher than cells with no zVAD.
http://www.pnas.org/content/pnas/111/31/E3206.full.pdf
You can still normalize your data to your untreated cells. This is totally acceptable. The real comparison in your case would be between your candidate necroptosis inhibitor and Nec-1 and how well the candidate inhibits necroptosis when compared to Nec-1.
If you really want to equalize OD values in your untreated/DMSO treated cells and TNF + Nec-1 cells, try adding Nec-1 to your untreated/DMSO cells to prevent any low level cell death.
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