I am using LDH assay to measure cell cytotoxicity of N27 cells treated with MPP+. N27 cells have been treated with 160 µM, 320 µM, 640 µM, 1.2 mM, and 2.5 mM of MPP+. After 24 hour incubation cells are not dying. When I treat cells with 100 µM, 200 µM, and 400 µM of H2O2, N27 cells are dead after 24 hours.
Initially we thought that MPP+ was the problem, we order new MPP+ and solubilized MPP+ in water, stored aliqouts in -20 celcius. Still did not see cell death for N27 cells.
We recently order new batch of cells and I treated those new batch of cells with mpp+, and still no cell death for N27 cells.
Does anyone have an established protocol I can follow to induce cell death in N27 cells using MPP+?
Hi,
are you restricted to LDH as endpoint for cytotoxicity? Remember that it measures membrane integrity, which may still be uncompromized following the 24h treatment with MPP+, which interferes with energy metabolism. You might want to try a different cytotoxicity test with an endpoint more closely related to your substance's mode of action, e.g. MTT (simplified measuring cellular respiration), Neutral red uptake (indirectly influenced by ATP contents) or direct ATP measurement, which in my opinion would be the most appropriate approach for your substance.
Best regards
Hi,
are you restricted to LDH as endpoint for cytotoxicity? Remember that it measures membrane integrity, which may still be uncompromized following the 24h treatment with MPP+, which interferes with energy metabolism. You might want to try a different cytotoxicity test with an endpoint more closely related to your substance's mode of action, e.g. MTT (simplified measuring cellular respiration), Neutral red uptake (indirectly influenced by ATP contents) or direct ATP measurement, which in my opinion would be the most appropriate approach for your substance.
Best regards
According to one literature ref (Toxicol Appl Pharmacol. 2007 May 1;220(3):341-8. Epub 2007 Feb 12.) you should try 48 hr. The authors of the above paper saw little cell death (by LDH assay) at 3 mM and 24 hr.
Hi Dionne,
for easiness/cheapness you may also consider trypan blue staining, which relies on the same principle as the LDH-test (membrane damage): just to *see* what happened to your cells. The other easy test would be the already recommended neutral red uptake.
Kind regards
Peter
Hi Dionne,
Completely agree with the previous contributors.
Judging by the MOA of MPP+, either MTT (or any of its "spin-offs") measuring mitochondrial activity, or a neutral red (NR) cytotoxicity/viability assay, should be good alternatives. Direct ATP measurement assays (and kits) are also widely available.
You may also consider reducing serum concentration in your assay medium. MPP+ is a highly positively charged molecule and I would expect it to bind to serum proteins.
Good luck and best wishes!
Erik
Hi Dionne,
The original N27 cells may have lost dopaminergic phenotype after repeated passaging. We have done single cell cloning from the original cell line to create N27A cell line which has nearly every cell expresses TH and DAT. Cells have a neuronal appearance and grow well. The new N27A cells are more sensitive to MPP+ than the original N27 cells. You can find the details in our newly published paper: http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0160847
We can provide the new N27A cells to research community if you are interested.
Wenbo Zhou, PhD
Dear Dionne,
I agree with Dr. Zhou on this. We were able to sensitize a different cell line (C17.2) to MPP+ by overexpressing DAT gene in these previously insensitive cells. Michael
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