I regularly use LDH and MTT assays together for investigating nanoparticle toxicity. I find the MTT assay more reliable, as I have found nanoparticle toxicity can manifest by interfering with attachment of cells to 96 well growth surfaces. Therefore you get reduced assay signal with LDH, purely because there are fewer cells. If your gold nanorods are toxic at all concentrations, you may get detachment of cells reducing LDH signal to baseline for all concentrations - just a thought, as I do not know how cytotoxic gold nanorods would be on your cell line? Alternatively, there is plenty of literature with nanoparticles, particularly highly refractive materials such a nanogold, interfering with cytotoxicity assays in other ways (such as optical interference). Try some of the literature attached to see if you can find some answers to your problem. Good luck!

Probably this might be the reason, you might not have reached the minimum toxic concentration of nanorods....try from 1nM to 10nM.

The LDH assay shows the cell membrane damage, it might be possible that the metabolic activity is reduced but the cell membrane is not damaged drastically. However, since you have incubated your cells with nano particles for 7 days and the MTT assay is showing high toxicity, you may need to check your protocole.

Marc, I did not treat the supernatant. I centrifuged the plates and removed the supernatent in another plate and followed the ldh protocol by sigma aldrich

Ventakachalam, from the MTT assay i performed these concentrations are toxic to the 3T3's

Interesting - I have seen similiar responses with other NPs effect on cell viability assays. It seems plausible the the Au nanorods are potent inhibitors of LDH or possibly interfere with electron transfer to NAD(P)H. Are you measuring this spectrophotometrically @ 450nm? Any appreciable absorption of the Au nanorad at this wavelength?

Have you thought about using Trypan Blue exclusion as an alternate assay?

Some nanoparticles, particularly at higher concentrations do interfere with these assays. Proper controlsand blanks such as using particle with the medium only may be needed.

Yasaman,

this is the protocol i am following

http://www.sigmaaldrich.com/etc/medialib/docs/Sigma/Bulletin/tox7bul.Par.0001.File.tmp/tox7bul.pdf

Wayne, I am mesuring the background absorbance at 690 and taking the ldh readings at 490.

If i am right,Trypan blue exclusion will help me get cell viabilty which i did get from MTT that i performed. i wanna perform ldh for membrane integrity.

Navin, Thanks

Mark, I am performing all these procedures in a 96 well plate. But I will try adding the centrifugation step after the supernatent is removed.

Could it b possibly MTT itself is toxic? Did u try running MTT and media together without cells and MTT , cells with media without nanoparticles? Also.. how about trying some other vital stain like XTT to counter check..

trypan blue exclusion and LDH both measure membrane integrity, while MTT measures metabolism as somebody here already mentioned.

I would suggest to check for interference of gold rods with the assay, by comparing positive control sups alone (ie sups of lyzed cells, containing lots of LDH) with positive control where ýou add the highest titer of gold rods AFTER the treatment. thus you will see whether the gold rods interfer with the colour development in the assay, if so you can start with centrifugation steps etc to get rid of them.

Good luck.

I regularly use LDH and MTT assays together for investigating nanoparticle toxicity. I find the MTT assay more reliable, as I have found nanoparticle toxicity can manifest by interfering with attachment of cells to 96 well growth surfaces. Therefore you get reduced assay signal with LDH, purely because there are fewer cells. If your gold nanorods are toxic at all concentrations, you may get detachment of cells reducing LDH signal to baseline for all concentrations - just a thought, as I do not know how cytotoxic gold nanorods would be on your cell line? Alternatively, there is plenty of literature with nanoparticles, particularly highly refractive materials such a nanogold, interfering with cytotoxicity assays in other ways (such as optical interference). Try some of the literature attached to see if you can find some answers to your problem. Good luck!

Here's another paper that might be of use - sorry, I didn't realise it only allows one upload at a time here.

Mark - great references! Thanks

Kristina - good suggestion, that should expose any assay issues

Komal,

I have a suggestion for you and this is what I have done myself when analyzing the cytotoxicity of metallic particles using MTS and LDH assays.

For each of your conditions, consider a blank in which you will have exactly the same concentration of your particles without having your cells . Read the absorbance of the blank at 490 nm as well and subtract this background from the data you read for each condition. I think this gives you a better background subtraction than 690 nm and it will help you to figure out if your particles have any interference with the assay.

Most in vitro toxicity assays are based on the use of colorimetric or fluorescent dyes to determine cell viability or functionality. Although they are accepted as general toxicity detection methods, they could not be completely accurate when testing nanomaterials, due to nanoparticle-dye interaction or adsorption, nanoparticle color or autofluorescence. You should make sure you have washed well, so that you remove most nanoparticles before the assays. But anyway it is necessary to perform additional and more specific tests to assess nanoparticle toxicity.

Yes, NP will give varied results. see

Monteiro-Riviere NA, Inman AO, Zhang LW. Limitations and relative utility of screening assays to asses engineered nanoparticle toxicity in a human cell line. Toxicology and Applied Pharmacology 234: 222-235, 2009

One way to tell if your cells are actually dying or not, as you said the extreme toxicity obsereved with MTT, I'd suggest just looking at the morphology to get a general idea if your cells are actually 'dying/dead' due to the gold nanorods, or if they maintain their morphology and do not exhibit apototic or necrotic charateristics, such as enlarged nuclei, rounding up, changes in refractive index, membrane blebbeing, large amounts of floating cells or cell debris, or other obvious signs of cellular insult and death. There should be images of what your cell lines look like when dead and dying too, just check the literature. Also just try a simple trypan blue assay, by plating your cells in six well plates, dose with the concentrations of NPs you desribed and each day for 7 days do a viability count using Trypan Blue; be sure to have a control well of the cells for each day with no addition of anything except a media change if necessary, and also a control that includes your vehicle/solvent (e.g., PBS, DPBS, DMSO, media, etc; without the Gold nanorods) of which you have the NPs in when you dose. This will give you at least some indication if you are experincing cytotoxicity.

I think it is better to do the assay on the supernant. So, after you incubate GNRs (Au-rods) with cells, spin them down and do the assay on the supernant. Because, if your GNRs kill the cells, LDH enzyme should be released to the media so after you spin down the cells and the GNRs, you will only have the supernant with the enzyme to be assayed.

You can also still do the assay the way that you're doing you just need to have a control " Au-Rods + media ' so you can subtract that from your original sample.

I hope these suggestions are helpful.

Single-walled carbon nanotubes (SWCNT), fullerenes (C60), carbon black (CB), nC60, and quantum dots (QD)

have been studied in vitro to determine their toxicity in a number of cell types. Here, we report that classical

dye-based assays such as MTT and neutral red (NR) that determine cell viability produce invalid results with

some NM (nanomaterials) due to NM/dye interactions and/or NM adsorption of the dye/dye products. In this

study, human epidermal keratinocytes (HEK) were exposed in vitro to CB, SWCNT, C60, nC60, and QD to assess

viability with calcein AM (CAM), Live/Dead (LD), NR, MTT, Celltiter 96® AQueous One (96 AQ), alamar Blue

(aB), Celltiter-Blue® (CTB), CytoTox One™ (CTO), and flow cytometry. In addition, trypan blue (TB) was

quantitated by light microscopy. Assay linearity (R2 value) was determined with HEK plated at concentrations

from 0 to 25,000 cells per well in 96-well plates. HEK were treated with serial dilutions of each NM for 24 h

and assessed with each of the viability assays. TB, CAM and LD assays, which depend on direct staining of

living and/or dead cells, were difficult to interpret due to physical interference of the NM with cells. Results of

the dye-based assays varied a great deal, depending on the interactions of the dye/dye product with the

carbon nanomaterials (CNM). Results show the optimal high throughput assay for use with carbon and

noncarbon NM was 96 AQ. This study shows that, unlike small molecules, CNM interact with assay markers

to cause variable results with classical toxicology assays and may not be suitable for assessing nanoparticle

cytotoxicity. Therefore, more than one assay may be required when determining nanoparticle toxicity for risk

assessment.

© 2008 Elsevier Inc. All rights reserved.

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