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. 2021 May 10;22(9):5038. doi: 10.3390/ijms22095038

Table 1.

Advantages and limitations of in vitro assays to study general cytotoxicity.

Assay Advantages Limitations References
Membrane integrity LDH leakage
assay

  • Sensitivity, agility and low cost.

  • Multiple time points analysis in a single test run.

  • High stability of the LDH enzyme.

  • Interference of cell culture components/test chemical with the LDH stability.

 [9,10,11,12,13]
Calcein-AM
assay

  • Simplicity, safety and low cost.

  • Suitability for HTP strategies.

  • Possibility of combining with other read-outs in a single test run.

  • Spontaneous leakage of the dye.

  • Stability of the signal.

  • Limited dye uptake in certain cell types.

  • Potential signal overlap between the calcein and the test chemical.

 [14,15,16]
Protease activity
assay

  • Multiple time points analysis in a single test run.

  • Possibility of combining with other read-outs in a single test run.

  • Suitability for HTP strategies.

  • Interference of cell culture components with the protease activity.

 [17,18]
Trypan blue
exclusion assay

  • Agility, simplicity and low cost.

  • Intra-operator/inter-operator variability.

  • Dichotomic nature of the results.

  • Sensitivity can be compromised by the concentrations and exposure time to the dye.

 [19,20,21]
Mitochondrial functionality Tetrazolium salt
assays

  • Simplicity and reproducibility.

  • Low cost.

  • Variable results depending on the cell culture stage/cell type.

  • Cell culture components/test chemical can catalyse MTT reduction.

  • Potential cytotoxicity of the reagents.

  • Lytic endpoint methodology.

 [9,22,23,24,25,26]
Resazurin reduction assay

  • Agility, sensitivity and simplicity.

  • Possibility of combining with other read-outs in a single test run.

  • Suitability for HTP strategies.

  • Multiple time points analysis in a single test run.

  • Potential cytotoxicity of the reagents.

  • Optimisation for each cell type.

  • Interference of cell culture components with the colorimetric signal.

 [14,27,28,29,30,31,32]
ATP content
assay

  • Agility, sensitivity and reproducibility.

  • Stability of the signal.

  • Low background noise.

  • Detection of early cytotoxicity.

  • Applicable to evaluate 3D cultures.

  • Test chemical and/or cell culture conditions can alter luciferase activity.

  • Lytic endpoint methodology.

  • Levels of ATP can be compromise by ATPases present in the media.

  • Expensive.

 [10,27,33,34,35,36,37,38]
Mitochondrial
membrane potential evaluation:
fluorescent probe-based assays

  • Reliable indicator of mitochondrial functionality.

  • Multiplatform evaluation (flow cytometer, fluorescence microscope or plate reader).

  • Low sensitivity and non-specificity of certain probes.

  • Potential cytotoxicity of the probes.

  • Requires the use of pharmacological controls and/or complementary probes.

 [39,40,41,42,43]
Oxidative stress DCFH2-DA
fluorescence probe-based assay

  • Agility.

  • Multiplatform evaluation (flow cytometer, fluorescence microscope or plate reader).

  • Low sensitivity and non-specificity of certain probes.

  • Artificial amplification of the signal.

  • Spontaneous leakage of certain probes.

 [44,45,46,47,48,49,50,51,52]
DHE/Mito-HE fluorescence probe-based assays

  • Agility.

  • Multiplatform evaluation (flow cytometer, fluorescence microscope or plate reader).

  • Evaluation of mitochondrial ROS levels.

  • Low sensitivity and non-specificity of the probe.

  • Optimisation of the probe concentration.

 [53,54,55,56,57]
Lipid peroxidation: MDA/TBARS assay

  • Agility, simplicity, low cost.

  • Low specificity, artificial amplification of the signal.

  • Relatively low detection limit.

 [58,59,60,61,62]
Enzymatic
antioxidants activity assays

  • Specificity.

  • Quantitative and functional nature of the results.

  • Lytic endpoint methodology.

  • No information about cellular localisation.

  • Requires the use of control conditions for isoform signatures.

 [63,64,65]
Cell death Annexin V
staining assay

  • Sensitivity.

  • Multiplatform evaluation (flow cytometer, fluorescence microscope).

  • Possibility of combining with other read-outs in a single test run.

  • Application in tissues and whole-body level.

  • Background signal.

  • Requirement for calcium ions in mM concentrations.

 [66,67,68,69,70,71,72]
PI dye uptake
assay

  • Versatility and low cost.

  • Multiplatform evaluation (flow cytometer, fluorescence microscope).

  • Possibility of combining with other read-outs in a single test run.

  • Suitability for HTP strategies.

  • Optimisation of the dye concentration, incubation time and washing steps.

  • Potentially mutagenic effect of the dye.

 [69,73,74]
Caspase activity
assays

  • Agility, reproducibility and sensitivity.

  • Multiplatform evaluation (flow and laser scanning cytometer).

  • Possibility of combining with other read-outs in a single test run.

  • Non-specific for a particular caspase.

  • Lytic endpoint methodology.

 [75,76,77,78,79]
TUNEL assay

  • Agility, sensitivity and simplicity.

  • Detection of early stages of apoptosis.

  • Multiplatform evaluation (light and fluorescence microscope, flow cytometer).

  • Possibility of combining with other read-outs in a single test run.

  • Expensive.

  • Time-consuming.

  • Subjected to false positive results.

 [68,80,81,82,83,84]
Miscellaneous Neutral red uptake
assay

  • Versatility, sensitivity and low cost.

  • Stability of the signal.

  • Suitability for HTP strategies.

  • Optimisation of the dye concentration and incubation time.

  • Impact of the test chemical on the dye activity.

  • Lytic endpoint methodology.

 [85,86,87,88,89,90,91,92,93,94,95]

ATP: adenosine triphosphate; Calcein-AM: calcein-acetoxymethyl; DCFH2-DA: 2′,7′-dichlorodihydrofluorescein diacetate; DHE: dihydroethidium; HTP: high-throughput; LDH: lactate dehydrogenase; MDA: malondialdehyde; MTT: 5-(3-carboxymethoxyphenyl)-2-(4,5-dimethylthiazoly)-3-(4-sulfophenyl)-tetrazolium; PI: propidium iodide; ROS: reactive oxygen species; TBARS: thiobarbituric acid reactive substance; TUNEL: terminal deoxynUcleotidyl transferase dUTP Nick End Labeling.