LDH-Glo™ Cytotoxicity Assay
- Overview
- Protocols
- Specifications
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Monitor Cytotoxicity from Small Numbers of Cells
The LDH-Glo™ Cytotoxicity Assay is a bioluminescent plate-based assay for quantifying lactate dehydrogenase (LDH) release into the culture medium upon plasma membrane damage. The bioluminescent detection is more sensitive than colorimetric or fluorescent methods, allowing accurate detection of LDH from a small number of cells, including primary cells and 3D cell cultures.
The assay involves removing only a small amount of cell media (2–5µl) from each treated well, allowing you to get more data by sampling the same well over time, and by using the remaining media and cells for other cell-based assays.
How the Assay Works
LDH released from damaged cells catalyzes the oxidation of lactate with concomitant reduction of NAD+ to NADH. Reductase uses NADH and reductase substrate to generate luciferin, which is converted to a bioluminescent signal by Ultra-Glo™ rLuciferase. The luminescent signal generated is proportional to the amount of LDH present.
Monitor Cytotoxicity in 3D Cell Cultures
The LDH-Glo™ Cytotoxicity Assay is well-suited for measuring LDH released from small numbers of membrane-damaged cells. Here is an example of measuring drug-induced toxicity in HCT116 spheroids. The time-dependent toxicity measurements were performed by repeatedly sampling media from the same wells.
HCT116 spheroids were formed using 2,500 cells/well in Corning 384-well Ultra-Low Attachment plates and treated with Doxorubicin. 2.5µl samples were collected from the same wells at the indicated time points and frozen in LDH Storage Buffer at a 1:10 dilution. Samples were thawed and further diluted 2.5-fold in LDH Storage Buffer before measuring LDH levels.See more data: Learn how this assay is used with different model systems, including SKBR3 cells to assess an antibody drug conjugate and in 3D human liver microtissues to monitor cytotoxicity over time, in the poster: New Sensitive Luminescent LDH-release Assay that Enables “Real-Time” Sampling from Individual 3D Microtissues, presented at SLAS Europe 2018.Detect Target Cell Cytotoxicity in ADCC Assays
The LDH-Glo™ Cytotoxicity assay can be used to assess cell death due to many mechanisms of action of biologics treatments, including complement dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-drug conjugate cytotoxicity. In this example, the LDH-Glo™ Assay was used to detect ADCC killing of target cells using the biologic rituximab.
Cells were plated at a 20:1 Effector (PBMC):Target (Daudi) ratio and treated with varying concentrations of rituximab (RTX) in triplicate for 4 or 6 hours. Medium samples (2.5µl) from the same wells were removed and diluted into LDH Storage Buffer (47.5µl) and frozen. 25µl of thawed samples were added to an equal volume of LDH Detection Reagent and luminescence was recorded after 60 minutes incubation at room temperature. Concentration of rituximab versus RLU was plotted and data was fit to 4PL curve to calculate EC50. 
RLU values from control wells (performed on the same plate as the rituximab treatment) indicate that there is minimal luminescent signal from target or effector cells alone, and that addition of rituximab is required for cell killing. ADCC = Effector cells +Target cells + RTX. RLU = Relative Luminescence Units. -
Protocols
Complete Protocol
LDH-Glo™ Cytotoxicity Assay Technical Manual
PDF (1 MB)
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Specifications
What's in the box?
Item Part # Size Reductase Substrate
G885A 1 × 55μl Lactate Dehydrogenase
J195A 1 × 1 each LDH Detection Enzyme Mix
J238A 1 × 10ml Certificate of Analysis
See all certificates for J2380Use Restrictions
For Research Use Only. Not for Use in Diagnostic Procedures.Storage Conditions
