Mar 04, 2025

Public workspaceQuantifying cell viability via LDH cytotoxicity assay

DOI
dx.doi.org/10.17504/protocols.io.3byl4wd62vo5/v1
  • 1Arcadia Science
  • Arcadia Science
Arcadia Science
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DOI: dx.doi.org/10.17504/protocols.io.3byl4wd62vo5/v1
Protocol Citation: Justin Donnelly 2025. Quantifying cell viability via LDH cytotoxicity assay. protocols.io https://dx.doi.org/10.17504/protocols.io.3byl4wd62vo5/v1
Manuscript citation:
Borges AL, Donnelly J, Morazan E, Rollins M. (2025). Compound 48/80 is toxic in HMC1.2 and RBL-2H3 cells. https://doi.org/10.57844/arcadia-3207-4695
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: January 24, 2025
Last Modified: March 13, 2025
Protocol Integer ID: 119041
Keywords: mast cell, basophil, cytotoxicity, LDH, enzyme assay
Abstract
This protocol describes a procedure to assess the cytotoxicity of in vitro treatments by measuring the level of lactate dehydrogenase (LDH) in cell culture supernatants via an enzyme activity assay. LDH is a cytosolic enzyme released into the supernatant upon cell lysis. This protocol uses the Abcam LDH assay kit (ab102526), which measures the increase in NADH produced by the released LDH enzyme over time.
Materials
ReagentLDH Assay Kit (Cytotoxicity)AbcamCatalog #ab65393
Black 96-well microtiter plate
Plate reader
1.5 mL microcentrifuge tubes
15 mL centrifuge tubes
Cell culture supernatants (analyte)
Protocol materials
ReagentLDH Assay Kit (Cytotoxicity)AbcamCatalog #ab65393
Materials, Step 1
Reagent preparation
Reagent preparation
10m
10m
This protocol uses the ReagentLDH Assay Kit (Cytotoxicity)AbcamCatalog #ab65393 . If you've already prepared kit reagents, skip to "Standard and substrate preparation."
Reconstitute NADH standard in Amount0.4 mL ultrapure water to generate a Concentration1.25 millimolar (mM) solution. Aliquot in Amount100 µL aliquots. Store remaining aliquots at Temperature-20 °C for future use.

Reconstitute recombinant LDH positive control in Amount200 µL LDH assay buffer. Aliquot in Amount20 µL aliquots. Store remaining aliquots at Temperature-20 °C for future use.

Reconstitute substrate mix in Amount1.1 mL ultrapure water. Allow to mix for ≥ Duration00:10:00 , vortexing intermittently. Aliquot in Amount185 µL aliquots. Store remaining aliquots at Temperature-20 °C for future use.

10m
Standard and substrate preparation
Standard and substrate preparation
Thaw LDH positive control on ice. Dilute entire aliquot to Amount200 µL in LDH assay buffer.

Thaw Concentration1.25 millimolar (mM) NADH standard aliquot on ice. Prepare standards as follows:
ABC
Volume NADH stock (µL)Volume assay buffer (µL)[NADH] in well (µM)
01250
512050
10115100
15110150
20105200
25100250

Thaw LDH substrate aliquot on ice. Dilute 25× substrate to 1× in LDH assay buffer. Vortex to mix. Prepare ≥ Amount50 µL reaction mix per reaction (not including blank).

Sample preparation and analysis
Sample preparation and analysis
32m
32m
Analyze samples in technical triplicate. For each sample, prepare appropriate dilutions in LDH assay buffer in appropriate wells of a black, 96-well microtiter plate such that each well has a total volume of
Amount50 µL . Depending on assay setup, you may need to optimize supernatant dilution factor.
Note
As an absorbance assay, the dynamic range of the LDH assay is limited. Ideally, the detected concentrations of NADH (which we use to calculate LDH activity) should fall within the range of the standards (50–250 µM). You may need to dilute analyte samples to achieve this.

In our hands, we found that no dilution was necessary for samples that didn't have significant supernatant LDH activity (i.e., when treatment had little impact on viability). For samples with significant LDH activity, a 5× dilution was generally appropriate. When testing unknown samples, we recommend analyzing both 1× and 5× dilutions.


Add standards (prepared in step 6) and controls to appropriate wells. Standards and controls are detailed in the following table:
ABC
ControlVolume per well (µL)Technical replicates
NADH standards50Duplicate
LDH (1× dilution)50Triplicate
LDH (5× dilution)10 (+ 40 µL buffer)Triplicate
Blank100Triplicate

Add Amount50 µL of reaction mix to all wells except the blank.

Measure absorbance at 450 nm on plate reader set to Temperature37 °C in kinetic mode, taking readings every Duration00:00:00 for a total of Duration00:30:00 .

32m