LDH cytotoxicity assay - help please - (Feb/06/2013 )

Dear all,

I am trying to assess cytotoxicity of suspension neutrophils by LDH release into the supernatant after various treatments. The neutrophils are at 10^6/ml and at the end of the assay I pellet them, remove the supernatant and use it to measure a number of things.

I found this homemade protocol on the internet on OPS Diagnostics website. I bought the reagents and prepared them exactly as stated. The only difference from the protocol is that I added the samples to the wells, then quickly mixed the Tris, Li lactate and PMS/INT/NAD and added them to the samples. I can't believe this makes any difference. Unfortunately I do not see any real colour change - even the positive control of 10^6 fully lysed cells barely causes any OD change. If I leave the plate for longer there is a slight shift in OD in all wells, but this may be the solution darkening naturally once mixed. I think the protocol below is designed to test efficiency of lysis rather than relatively small levels of lysis ( I am hoping to show little if any cytotoxicity) so perhaps it is insufficiently sensitive to pick up any LDH released in my samples, even in the positive control.

Does anyone have any experience with this assay - specifically can you suggest changes to the concentrations of the various reagents which may be more sensitive? Or do you have a different protocol for which I can use the reagents already purchased. There are obviously a lot of LDH assay kits on the market - about £230 for 500 wells but these seem to rely on the cells being plated on the 96 well plate rather than simply using supernatant. Any experience with these kits?

Thanks in advance for any help and advice.

Jim

Lactate Dehydrogenase Protocol
Lactate dehydrogenase is an enzyme which is useful for monitoring cell death and disruption. Kits are available to run LDH assays from many biological suppliers. Alternatively, the assay can be developed in-house more cost effectively. The following protocol is for an in-house recipe. LDH assays are used routinely to assess the efficiency of homogenization methods in our product development lab. For more information on OPS Diagnostics' homogenization products, click here.

200 mM TRIS, pH 8

Most LDH works well at this pH. To prepare, add 22.2 gm of Tris-HCl (Sigma-Aldrich, T-3253 or equivalent) and 10.6 gm of Tris-base (Sigma-Aldrich, T4661 or equivalent) to 1 L water.
50 mM Li Lactate
Prepare by adding 49 mg lithium lactate (Sigma L-1500 or equivalent) into 2.5 ml of water.
NAD/PMS/INT Solution
Prepare the PMS, INT, NAD solution shortly before use. Dissolve INT (Sigma I-8377 or equivalent) into DMSO (Sigma D-8779 or equivalent) at a concentration of 3.3 mg/100 ul DMSO. Larger quantities of this solution can be made, aliquoted, and stored at -20°C (freezer).

Prepare a stock of Phenazine Methosulfate (Sigma P-9625), i.e., PMS, by dissolving 0.9 mg into 100 ul water. Larger volumes can be prepared, aliquoted and stored at -20°C.

Prepare NAD (Sigma N-0632 or equivalent) by adding 8.6 mg NAD to 2.3 ml water. More of this solution can also be made, aliquoted, and stored at -20°C.

Mix 100 ul PMS, 100 ul INT, and 2.3 ml NAD solution. This solution is good for several hours though it gradually darkens. Extra solution can be frozen at -20˚C.
Assay Protocol
The following assay is for a microplate reader, though it can be scaled up for cuvettes or tubes. A good wavelength for measuring OD is 490 nm. Wells should be prepared for all samples, ran in triplicate, plus three negative controls using water. If LDH is available, three positive controls can be ran as well.
1. Add all the reagents first, followed by the cell lysates. Do not cross contaminate the wells.
200 mM Tris, pH 8 50 ul
50 mM Li Lactate 50 ul
PMS, INT, NAD 50 ul
2. Add 50 ul sample or control. The assay can be run as an end point assay (wait 5 minutes and read the plate) or kinetic assay based on the microplate reader. Most accurate measurements determine the maximum slope during a kinetic assay.

-jimmacfarlane-

what is the supernatant? the culture media?

-Curtis-

Yep it's IMDM (no phenol red)

-jimmacfarlane-

I don't understand, the protocol is asking you to add the reagents to cell lysate, but you are using the supernatant for your readings? I'm not sure if that will work.

-Curtis-

Indeed, I think in retrospect that is the issue - their protocol is to test efficiency of cell lysis/homogenisation ie there is likely to be a large LDH release and the assay is relatively insensitive. I am trying to show that the treatments on my cells have low levels of cytotoxicity (ie other cellular contents released extracellularly in response to my treatments that I am able to measure are not simply due to membrane rupture and cell lysis). The LDH release is therefore expressed as a % of the release from the same number of fully lysed cells (using HCTA or Triton etc). So technically it should work with the supernatant but as I get almost no OD change even with the positive control (of the fully lysed 10^6 cells ) it suggests to me that the assay is too insensitive to pick up such dilute LDH. Hope that makes sense - I was wondering whether there is a way of altering the concentration of the various chemicals to make the assay more sensitiev and reproducible. (I understand it is a redox reaction involving NAD to NADH).

Thanks Jim

Seems odd that this isn't done more commonly but perhaps a dated assay now - folk at my uni have suggested just buying a kit.

-jimmacfarlane-

you know, i just worry about the volume of the media. if you decide to go ahead with your own method, which is using the media, then you must realize even if you have secretion into media your test should be really sensitive to give you any reading. i mean to me it's like you are using a diluted sample and the machine can't detect anything. i'm not sure about your positive controls though...

i work with a virus. my cells produce the virus and release it into media. if i use the supernatant for reading or other test it won't show much activity, but we centrifuge the media at high speed and purify the virus and run tests on that. but in your case it is more complicated. i hope you understand what im trying to say.

-Curtis-

Thanks for the help - it does make sense. It's useful that you are concluding the same thing as me - that the LDH is so dilute that it cannot be picked up in the assay. Yes it is a bit strange that the positive control doesn't work. It's a technique we use loads to test for products like superoxide in the supernatant (via a colorimetric assay) and also for cytokine production but these are picked up by sandwich elisa.

Will investigate the kits, but might have the same problem there!

Thanks Jim

-jimmacfarlane-

good luck

-Curtis-