We have found similar problems and encountered two types of problems. The first is the fact that during the MTT assay, the NM remain in assay solution and can contribute the adsorbance data. This can be easily corrected by performing the same assay in wells without cells and subtracting this 'background' adsorbance. Second, the MTT dye tends to 'stick' to certain NMs. This problem can also be solved, by first rinsing your cells with fresh media, thereby removing the NMs. We have also used WST-1 assays (Roche). In this assay, the culture medium with NMs is removed before adding the WST-1 assay solution, again solving the problems of interference. Have you seen this: Worle-Knirsch, J. M., et al. (2006) Nano Letters 6, 1261?

do you have a well with NM only. alternative is use cell viability kit from dojindo, which uses WST-8 to determine the cell viablity/death

media with cells, media without cells+ NMs diff. conc, Cells +NMs diff conc, tnxs for your suggestion, we will workout on your idea

It is also in accordance with ISO 10993-5:2009 to use a protein assay, e.g. BCA, for assessment of cell proliferation inhibition. The higher the (external/secreted) protein the higher is the proliferation inhibition.If there is an interference between the BCA assay and the nanomaterials can easily be tested by setting up a calibration set of BSA in presence of NM. At least, it may be worth a try.

Regards,

Alec

How are you taking your absorbance? In situ? Normally the dye is solubilised and then the OD measured in a separate plate. I can not visualise how the NMs can interfere if the test is done this way, unless it is the leached out chemicals that are your concerns.

If your nanomaterials are your concern while measuring abosorbance in MTT assay, Switch to Sulphorhodamine-B assay where you precipitate only the cellular proteins in the first step of the assay and is equally sensitive as MTT or WST.

I think you might want to determine the absorbance of your NM.

In case if the absorbance between NM and formazan at 570nm is very different, you can possibly do a row of serial dilution on your NM without cell. By this, you can minus the readings produce by your NM at that specific concentration.

If the difference between absorbance of NM and formazan is very little. Using the method above could be quite erroneous. Alternatively you can try other viability assay, such as LDH cytotoxciy assay whereby quantification of its activity is at 450 nm.

We have found similar problems and encountered two types of problems. The first is the fact that during the MTT assay, the NM remain in assay solution and can contribute the adsorbance data. This can be easily corrected by performing the same assay in wells without cells and subtracting this 'background' adsorbance. Second, the MTT dye tends to 'stick' to certain NMs. This problem can also be solved, by first rinsing your cells with fresh media, thereby removing the NMs. We have also used WST-1 assays (Roche). In this assay, the culture medium with NMs is removed before adding the WST-1 assay solution, again solving the problems of interference. Have you seen this: Worle-Knirsch, J. M., et al. (2006) Nano Letters 6, 1261?

thanks Lars for your valuable answer.

You should consider using the soluble MTT analogue WST instead of MTT as well.

You can use your NMs with all of the components such as MTT, medium, SDS etc without cell and get the absorbance measured and you can nullify it from the OD of the sample. Make ensure that the blank titer plate also has its own absorbance value which also you need to consider in your calculation. I would be happy if the information was helpful to you.

do a wavelenght scan for your NMs, also if your NMs are charged they are going to adsorb the dye. what is the concentration of NM's in the cell culture conditions. other factors include wettablity and reflection of the light during the reading of the plate may lead to the spurious results.

yes, when you are taking readings, the nanomaterials do interefere if they are at high concentrations. if you loose the lower concentrations it would have minimized the effects, while doing so, you can always measure the concentration of the nanoparticles separately in 96 well plates and the subtract from the results.

let me know if you have nay query!