Mar 19, 2026
Quantification of Cell Death and Cytotoxicity Using LDH-Glo™ Cytotoxicity Assay
- Elena Coccia1
- 1Icahn School of Medicine at Mount Sinai
Protocol Citation: Elena Coccia 2026. Quantification of Cell Death and Cytotoxicity Using LDH-Glo™ Cytotoxicity Assay. protocols.io https://dx.doi.org/10.17504/protocols.io.q26g776b3gwz/v1
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: February 12, 2026
Last Modified: March 19, 2026
Protocol Integer ID: 243131
Keywords: cell death, cytotoxicity, LDH-Glo™ Assay, lactate dehydrogenase, quantification of cell death, using ldh, cytotoxicity in cultured cell, ldh, damaged cell, cultured cell
Abstract
To quantify cell death and cytotoxicity in cultured cells using the LDH-Glo™ Cytotoxicity Assay, which measures lactate dehydrogenase (LDH) released from damaged cells.
Materials
Conditioned Media (Cell culture media (e.g., RPMI 1640)) -
Storage Buffer (200 mM Tris-HCl pH 7.3, 10% glycerol, 1% BSA)
LDH-Glo™ Assay Kit (Promega, #J2380) -
96-well Plate (Clear flat-bottom for luminescence measurement)
Reagents:
Buffer Tris-HCl - Buffer used for sample dilution and storage
Glycerol Glycerol - Stabilizer for LDH in storage buffer
BSA Bovine Serum Albumin - Protein used to stabilize LDH in storage buffer
Troubleshooting
Problem
Low luminescence signal
Solution
Ensure proper mixing of samples and detection reagent; check for reagent expiration.
Problem
High background signal
Solution
Verify that the assay plate is free from contamination; ensure proper controls are included.
Safety warnings
Handle all reagents and samples with care, using appropriate personal protective equipment (PPE). Dispose of all waste according to institutional biosafety guidelines.
Sample Collection and Storage
Harvest conditioned media from cell cultures.
Sample Preparation
1. Collect 2 μL of conditioned media from each well. 2. Dilute the collected media in 298 μL of storage buffer (200 mM Tris-HCl pH 7.3, 10% glycerol, 1% BSA). 3. Store samples at -20 °C until analysis.
LDH-Glo™ Assay Execution
Perform the assay according to the manufacturer's instructions.
Assay Setup
1. Thaw stored samples and mix gently. 2. Transfer 50 μL of each diluted sample into a 96-well plate. 3. Add 50 μL of LDH detection reagent to each well (1:1 ratio). 4. Incubate the plate at room temperature for 30 minutes.
Luminescence Measurement
1. Measure luminescence using a plate reader.
Protocol references