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. 

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 µl 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 µl 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 µl PMS, 100 µl 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.
    2. Reagents
    200 mM TRIS, pH 8 50 µl
    50 mM Lithium Lactate 50 µl
    PMS, INT, NAD 50 µl
  2.   Add 50 µl 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.

    3. An example of using the LDH assay for measuring homogenization efficiency can be found at the following link, Homogenization of Mouse Muscle: A Comparison of Methods.

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