LDH-Blue™
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Cat.code:
rep-ldh-1NEW
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ABOUT
Colorimetric LDH release assay for cell death detection
LDH-Blue™ is a high-performance, colorimetric assay for measuring lactate dehydrogenase (LDH) activity in damaged cell supernatant, providing a reliable readout of cell death and a powerful complement to traditional cell viability assays. Designed for simplicity and sensitivity, LDH-Blue™ provides a robust readout of lytic cell death, including necroptosis, pyroptosis, ferroptosis, late apoptosis, and immune cell-mediated cytotoxicity.
Assay principle
LDH is a stable cytoplasmic enzyme released into the culture medium when the plasma membrane is compromised. LDH-Blue™ detects extracellular LDH via a coupled enzymatic reaction that converts a tetrazolium salt into a blue formazan product, proportional to LDH activity. This color change can be quantified using a spectrophotometer at 650 nm.
Key features
- Practical format: Add, Mix, & Read
- Visual results in < 30 min
- Precise and sensitive
- Absorbance linearity with the number of lysed cells
- No interference with phenol red
- Cost-efficient
All InvivoGen products are for internal research use only, and not for human or veterinary use.
SPECIFICATIONS
Specifications
50 µl (if using P96 well plates)
Each lot is functionally tested and validated.
CONTENTS
Contents
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Product:LDH-Blue™
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Cat code:rep-ldh-1
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Quantity:200 tests
- 1 vial of Reagent Mix (lyophilized)
- 1 bottle (10 ml) Assay Buffer
- 1 bottle (2.5 ml) Lysis Buffer
- 1 bottle (10 ml) Stop Solution
Each vial of Reagent Mix is sufficient for monitoring LDH activity in 2 x 96-well plates (~200 tests).
Shipping & Storage
- Shipping method: Room temperature
- Store the Reagent Mix at -20°C
- Store the Assay Buffer, Lysis Buffer, and Stop Solution at 4°C
- Avoid repeated freeze-thaw cycles
- Protect from light
Storage:
Caution:
Details
Assay principle
The LDH activity is typically determined by utilizing a coupled enzymatic reaction where LDH oxidizes lactate to pyruvate, converting NAD+ to NADH. Subsequent reduction of tetrazolium salts by NADH and phenazine methosulfate (PMS) leads to the formation of colored formazan. The amount of formazan is directly proportional to cell viability. It can be easily detected with the naked eye and quantified using a spectrophotometer.
InvivoGen's LDH-Blue™ kit contains all the necessary compounds for assessing LDH activity, including the substrate which reduction produces blue formazan. This product can be quantified by measuring the absorbance at 650 nm.
DOCUMENTS
Documents
Technical Data Sheet
Validation Data Sheet
Safety Data Sheet
Certificate of analysis
Need a CoA ?
Powered by BiozFAQ
LDH-Blue™
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.
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.
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).
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.
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.
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.