CyQUANT Cytotoxicity Assays are simple and reliable assays that can measure chemical or cell-mediated cellular damage. CyQUANT LDH Cytotoxicity Assays and CyQUANT Cytotoxicity Assay Kit (G6PD Release Assay) use a colorimetric- or fluorescence-based detection method to measure the level of a cytosolic enzyme—LDH or G6PD—that is released into the media when cells are damaged. Optimized for use on microplate readers, these assays can be used to continuously monitor the health of cells over time since cell lysis is not required.
Selection guide for CyQUANT Cytotoxicity Assays
| CyQUANT LDH Cytotoxicity | CyQUANT LDH Cytotoxicity Assay, fluorescence | CyQUANT Cytotoxicity Assay Kit (G6PD Release Assay) | |
|---|---|---|---|
| Assay type | Colorimetric | Fluorescent | Colorimetric, Fluorescent |
| Use |
| ||
| Application |
|
| |
| Measurement | Lactate dehydrogenase (LDH) | Glucose 6-phosphate dehydrogenase (G6PD) | |
| Mechanism of detection | Tetrazolium salt (INT) is reduced to a red formazan product  | Resazurin is reduced to red-fluorescent resorufin | Resazurin is reduced to red-fluorescent resorufin |
| Incubation time | 30 min | 10 min | 30 min |
| Efflux detection reagent (Ex./Em.) | Formazan (Abs: 490 nm) | Resorufin (560/590 nm) | Resorufin (560/590 nm) |
| Cat. No. | C20300 | C20302 | V23111 |
CyQUANT LDH Cytotoxicity Assay Kits
Lactate dehydrogenase (LDH) is a cytosolic enzyme present in many different cell types. The release of LDH is ideal for research for screening cellular injury, tissue damage, and disease such as liver disease or heart failure. LDH can also be measured to monitor cancer treatment as many cancers can cause an increase in LDH levels (1,2).
When the plasma membrane of cells is damaged, LDH is released into the surrounding medium. The Thermo Fisher Scientific CyQUANT LDH Cytotoxicity Assay Kits are reliable colorimetric or fluorescent assays that can be used to quantitatively measure LDH released into the media from damaged cells.
CyQUANT LDH cytotoxicity assay
The CyQUANT LDH Cytotoxicity Assay is a simple and reliable colorimetric assay to measure cytotoxicity using tetrazolium salt (INT). LDH released from dead or dying cells converts lactate to pyruvate through the reduction of NAD+ to NADH. The resulting NADH reduces INT to a red formazan product that can be measured on a microplate reader at 490 nm (Figure 1). The amount of formazan generated is directly proportional to the amount of released LDH (Figure 2).
Figure 1. CyQUANT LDH Cytotoxicity Assay mechanism.
Figure 2. Determination of cytotoxicity with the CyQUANT LDH Cytotoxicity Assay. A549 cells were seeded into a 96-well plate using either MEM containing 10% FBS or MEM without serum. After an overnight incubation, the cells were treated with water (Spontaneous LDH Release) or 10X Lysis Solution (Maximum LDH Release). After treatment, the medium was removed and placed into a 96-well plate. The amount of LDH released into the medium was determined using the CyQUANT LDH Cytotoxicity Assay following manufacturer’s instructions. The amount of LDH released and detected correlated very well with the number of cells per well. Note: the endogenous LDH present in FBS results in higher background absorbance. Additional wells without cells, but containing complete medium with FBS, can be used as controls to help reduce the amount of LDH interference resulting from the serum.
Used alone or easily multiplexed, CyQUANT LDH Cytotoxicity Assay can be used to monitor cell health in drug-treated cells with other viability reagents. While LDH is used to measure cytotoxicity, resazurin-based alamarBlue HS and PrestoBlue HS are used to determine viability; an increase in LDH absorbance and a decrease in alamarBlue and PrestoBlue fluorescence are indicative of the cytotoxic effects from the drug-treatment (Figure 3).
Learn more about alamarBlue HS Cell Viability reagent
Learn more about PrestoBlue Cell Viability reagent
Figure 3. Multiplexing to simultaneously detect changes in cytotoxicity and viability. A549 cells were seeded and, after an overnight incubation, the cells were subjected to gambogic acid treatment. Post-treatment, a 50 µL aliquot of the medium was removed and transferred to a 96-well plate for LDH cellular cytotoxicity concentration determination. To determine viability, alamarBlue HS or PrestoBlue HS were added to the wells containing the treated cells and, following manufacturer’s instructions, a reduction of viability was detected with increasing concentration of gambogic acid. The amount of LDH in the aliquoted post-gambogic acid treated medium was determined by using the CyQUANT LDH Cytotoxicity Assay. An increase in LDH concentration representing an increase in cytotoxicity is displayed (left y-axis).
CyQUANT LDH Cytotoxicity Assay, fluorescence
The CyQUANT LDH Cytotoxicity Assay, fluorescence is a simple and reliable fluorescent assay to measure cytotoxicity using resazurin. During this process, LDH released from damaged cells converts lactate to pyruvate through the reduction of NAD+ to NADH. The resulting NADH reduces resazurin to fluorescent resorufin that can be quantified on a microplate reader using an excitation of 560 nm and emission of 590 nm (Figure 4). The amount of resorufin generated is directly proportional to the amount of released LDH and indicative of cytotoxicity (Figure 5).
Figure 4. CyQUANT LDH Cytotoxicity Assay, fluorescence mechanism.
Figure 5. Determination of cytotoxicity with CyQUANT LDH Cytotoxicity Assay, fluorescence. A549 cells were seeded into 96-well plates with MEM containing 10% FBS. After an overnight incubation, the complete medium in half of the wells was replaced with MEM only (no FBS). The cells in complete and non-serum media wells were treated with either water (Spontaneous LDH Release) or the 10X Lysis solution (Maximum LDH Release). After treatment, the medium was removed and placed into 96-well plates. The amount of LDH released into the medium was determined using the CyQUANT LDH Cytotoxicity Assay, fluorescence. The amount of LDH detected correlated very well (R2 values greater than 0.98) with the number of cells per well. Note: the endogenous LDH present in FBS results in higher background absorbance (left panel). Additional wells without cells, but containing complete medium including FBS, can be used as background controls to determine the amount of LDH interference resulting from the serum.
CyQUANT LDH Cytotoxicity Assay, fluorescence contains a highly purified resazurin reagent with a reduced amount of resorufin contamination compared to other resazurin-based reagents. An improved production process and formulation has resulted in a highly pure resazurin that helps improve signal-to-background and sensitivity over competitor LDH cytotoxicity assays (data generated in 2020) (Figure 6).
Figure 6. Comparison of fluorescence based LDH cytotoxicity assays. A549 cells were seeded into 96-well plates. After an overnight incubation, the cells were treated with various concentrations of gambogic acid. To determine changes in cytotoxicity, the CyQUANT LDH Cytotoxicity Assay, fluorescence, or the CytoTox-ONE™ Homogenous Membrane Integrity Assay (Promega) were used following manufacturer’s instructions. Both assays generated overlapping IC50 values, but the size of the dynamic range of the assay was 2-fold greater for the CyQUANT LDH Cytotoxicity Assay, fluorescence.
CyQUANT Cytotoxicity Assay Kit (G6PD Release Assay)
Glucose 6-phosphate dehydrogenase (G6PD) is an essential metabolic enzyme that is involved in many physiologic processes, including the pentose phosphate pathway. G6PD is involved in various biosynthetic functions, such as protecting against oxidative stress, fatty acid biosynthesis, and regulation cell growth and proliferation (3,4,5).
During cellular plasma membrane damage, G6PD is released into the surrounding medium. The Thermo Fisher Scientific CyQUANT Cytotoxicity Assay Kit (G6PD Release Assay) is a quick and efficient assay used to quantify cell death from the release of G6PD from cells. More sensitive than LDH assays, the G6PD release assay can detect as few as 500 cells, which is excellent for the analysis of low cell numbers.
The G6PD release cytotoxicity assay is a two-step enzymatic process that helps reduce resazurin into red-fluorescent resorufin. Oxidation of glucose 6-phosphate by G6PD generates NADPH which reduces resazurin to resorufin (Figure 7) that can be quantified on a microplate reader at an excitation/emission of 560/590 nm, with the fluorescent signal being beyond the autofluorescence of most biological samples. The resulting signal is proportional to the amount of G6PD released and correlates with the number of dead cells in the sample (Figure 8). The G6PD assay also has lower background signal since the G6PD levels in serum are lower than LDH levels, both of which are commonly used for cell culture (Figure 9).
Figure 7. CyQUANT Cytotoxicity Assay Kit (G6PD Release Assay) mechanism.
Figure 8. Detection of dead and dying cells using the CyQUANT Cytotoxicity Assay Kit (G6PD Release Assay). Jurkat cells were treated with 10 µM camptothecin for six hours, then assayed for glucose 6-phosphate dehydrogenase release using CyQUANT Cytotoxicity Kit (G6PD Release Assay). An untreated control sample is shown for comparison. The fluorescence was measured in a microplate reader (excitation/emission ~560/590 nm). A background of 55 fluorescence units was subtracted from each value.
Figure 9. Comparison of G6PD and LDH levels in serum. 10% bovine serum was assayed for the presence of lactate dehydrogenase (LDH, blue) and glucose 6-phosphate dehydrogenase (G6PD, red). G6PD was assayed using the CyQUANT Cytotoxicity Assay Kit (G6PD Release Assay); LDH was detected using a similar method, in which LDH reduces lactate to generate NADH using the CyQUANT LDH Cytotoxicity Assay, fluorescence. The result shows that, over the time course of the experiment, the serum generates a significantly lower signal in the G6PD assay than in the LDH assay, indicating G6PD is more sensitive in determining cellular cytotoxicity.
Expertly detect fluorescence with Thermo Scientific plate readers
High-sensitivity fluorescence detection for 6-1,536 samples can be quickly performed on the Varioskan ALF or Varioskan LUX Multimode Microplate Reader using Invitrogen reagents to enable optimal detection. Take advantage of automatic dynamic range selection to get optimal gain settings for each individual well and automation capabilities for even higher throughput.
Citations
- Chan FK, Moriwaki, K, De Rosa MJ (2013). Methods Mol Biol. 979:65-70. PMID: 23397389
- Farhana A, Lappin SL (2021). StatPearls [Internet]. PMID: 32491468
- Stanton RC (2012). IUBMB Life. 64(5):362-369. PMID: 22431005
- Yang H, Wu Y, Yen W, et al (2019) Cells. 8(9):1055. PMID: 31500396
- Tian W-N, Braunstein LD, Pang J, et al (1998) JBC. 273(17):P10609-10617. PMID: 9553122
Resources
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