LDH Cytotoxicity Assay
LDH-Blue™
One possible reason for the high variation in replicate values could be pipetting errors or insufficient mixing of the Reagent Mix Solution with supernatant samples. We recommend you repeat the experiment using another set of pipettes and/or mix the plate more thoroughly (avoiding bubble formation).
Since LDH-Blue™ is non-radioactive and uses absorbance-based detection, it can be multiplexed with fluorescence or luminescence-based assays from the same sample (with appropriate timing). For example, you can use LDH-Blue™ with InvivoGen's Lucia luciferase-based cellular assays: the HMGB1 release reporter cells THP1-HMGB1-Lucia™, the ferroptosis reporter cells HT1080-HMGB1-Lucia™, and the oxidative stress reporter cells HEK-Lucia-Star™ ARE.
To monitor cytotoxicity using InvivoGen's LDH-Blue™, you will need:
- test and control supernatants
- pipettes and pipette tips, including multi-channel pipette
- reagent reservoir
- flat-bottom transparent 96-well plates
- a spectrophotometer microplate reader capable of reading 650nm absorbance.
The kit components are shipped at room temperature. Store the Reagent Mix at -20 °C and protect it from light. Store the Assay Buffer, Lysis Buffer, and Stop Solution at +4 °C. When stored as directed, the kit is stable for up to 1 year from the date of receipt. Once resuspended, the Reagent Mix is stable for 1 week when stored at -20 °C.
LDH-Blue™ detects lytic cell death associated with plasma membrane disruption, including necroptosis, pyroptosis, ferroptosis, late apoptosis, and immune cell-mediated cytotoxicity. It is not specific to apoptotic pathways without membrane rupture.
LDH-Blue™ primarily detects cell death associated with membrane integrity loss, such as necrosis or late-stage apoptosis. For early apoptosis detection, complementary assays like Annexin V staining are recommended.
Absolutely. LDH-Blue™ is ideal for T cell, NK cell, or antibody-mediated cytotoxicity assays. Ensure proper controls for spontaneous and maximum release.
Yes, LDH-Blue™ is sensitive enough to detect LDH release from samples with low cell numbers, including 3D microtissues and primary cells. Its high sensitivity ensures accurate cytotoxicity assessment in various experimental setups.
Absolutely. LDH-Blue™ is designed for use with standard 96-well and 384-well plate formats, making it suitable for high-throughput screening of cytotoxicity in drug discovery and other large-scale studies. You must change the volumes of the supernatant and reagent mix solution (and stop solution) according to the plate format. For a 96-well plate, please see the technical datasheet.
Yes, LDH-Blue™ is optimized for use with serum-containing or serum-free media. Background levels may vary slightly with serum type.
Certain substances, such as high concentrations of reducing agents or components that affect enzyme activity, may interfere with the assay. It's advisable to include appropriate controls to account for potential interferences.
Depending on your assay, you may want to store the supernatants for later reading. We recommend storing the supernatant at 4°C for up to 48 hours and at -80°C for a longer period. Do not store at -20°C.
There are different possible explanations for low absorbance values:
- The LDH reaction time is too short. If so, do not use the optional Stop Solution and perform kinetic readings of LDH activity.
- The cell culture density is too low. If so, repeat the experiment to optimize cell number.
- The test compound may interfere with the LDH oxydo-reductive reaction (see FAQ above).
- Bubbles are present in the wells when performing the reading. Avoid bubble formation when pipetting and mixing.