CyQUANT Cytotoxicity Assays

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.

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.

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).

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.

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.

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.