Hello,
I've incubated MG63 with increasing concentrations of HA nanoparticles. After 24 hours I conducted an alamar blue assay and subsequently a Hoechst DNA assay.
After analysing the results, the alamar blue shows a decreasing trend in metabolic activity with increasing NP concentration. However after looking at the hoechst DNA assay I see an opposing trend with DNA content and hence relative cell number actually increasing with increasing NP concentration.
Could someone possibly shed light as to what is happening? Could it possibly be interference with NPs?
Thanks
A few things you might want to consider:
HA nanoparticles may interfere with alamar blue. Run control wells with ascorbic acid.
Do you aspirate your HA nanoparticles before adding either alamar blue or Hochest? If not then run a parallel assay where you remove the nanoparticles and wash your cells before adding alamar blue or Hoechst.
Are you doing colorimetric or fluorimetric measurements for alamar blue? Fluorimetric measurements are very sensitive and should be preferred if available.
Try to see the cells from both groups under light microscope. It may or may not help you but sometimes a simple thing like this might help.
Good luck
Hello Adeel, thank you for your advice. Its very much appreciated!
Yes the NPs were aspirated however i did notice that at higher concentrations of NP a film formed over the base of the wells which was not possible to remove without also removing the cells.
The fluorescence was read for each of the assays.
Thanks
I agree with Adeel Masood Butt.
Correct controls could shed more light on these findings.
Some additional hypothesis to test:
1. NPs may affect mitochondria efficiency and so decrease total metabolic activity.
2. Alamar blue could contains some small molecule electron carriers to enhance reduction of Rezazurin,so NPs could affects active concentration and/or trafficking of these carriers.
3. NPs could account to false positive signal during measuring of DNA content or increase quantum efficiency of hoechst:DNA complex.
I guess some of these ideas could be tested with mitochondrial potential dyes and some in cell free system.
Hope it would be helpful.
- Badges
- Science topic
- Similar topics
- Chemistry
- Nanotechnology
- Nanostructured Materials
- Nanoparticles