Table 2.

In vitro assays to evaluate toxicity of NP preparations.

Assay	Mechanism	Advantages/use	Limitations	References
Chromium-51 release assay	Detection of irreversible damage to cell plasma membrane.	High sensitivity, accuracy, and simplicity.	Requires handling of hazardous radioisotopes, and needs pre-labeling of cells.	[136–138]
LDH release assay	Detection of damage to cell membrane by measuring the release of intracellular LDH enzyme.	No need in cell labeling and use of isotopes.	Less sensitive, takes more time and less high-throughput than above.	[139,140]
Metabolic activity assay (MTT, MTS, etc.)	Colorimetric assay measuring cellular metabolic activity, such as ability to reduce tetrazolium dye.	Simple, Cost-effective, Indicates total metabolic status.	Numerous intracellular factors can influence reduction of dye leading to inaccurate results.	[141–144]
Protease activity assays (CytoTox-Glo)	Luminescent assay measuring activity of intracellular protease released from cells, which have lost their membrane integrity.	Different protease activities can be measured.	Costly, Signal interference from fluorescent nanoparticles.	[145–147]
Calcein AM assay	Calcein AM is a permeable molecule that enters cells and is hydrolyzed by intracellular estrases to Calcein which is fluorescent and is retained inside cells.	Simple, Indicative of membrane damage.	Signal interference from fluorescent nanoparticles.	[148–150]
Oxidative stress assays (DCFH assay)	Measures the release of reactive oxygen species by detecting conversion of substrates into fluorescent or colorimetric outputs.	Simple, Direct measure of cellular redox state.	Not a direct measure of H2O2.Several other oxidative species can oxidize DCFH. Released cytochrome C can oxidize DCFH.	[151–153]
Apoptosis assays (Annexin V staining, caspase activity assays)	Detects presence of phosphatidyl serine in apoptosing cells by annexin V staining; or measurement of caspases such as caspase 3 activated during apoptosis.	Simple, Distinguishes apoptosis from necrosis.	Annexin V staining requires live cells, and for adherent cells must use detachment agents that do not damage the cell membrane.	[154–156]
Genotoxicity assays (comet assay)	Single cell gel electrophoresis assay that measure DNA strand breaks.	Rapid, Indicates DNA damage.	Requires an internal reference to avoid variations. Inability to detect specific mutations generated.	[157–160]
Hemocompati bility assays (Hemolysis assay)	Spectrophotometric measurement of the amount of hemoglobin released.	Simple, Indicates RBC damage.	Nanoparticle binding to complement proteins can alter the hemolytic activity.	[161–164]
Macrophage cytokine profiling	Measures cytokines released from macrophages, using systems such as ELISA or Luminex bead array assays.	Simple, extensively used, High throughput assay for several cytokine measurement in one assay.	Endotoxin contamination can produce false positive results.	[165–167]
Leukocyte proliferation assay	Leukocytes are incubated with different concentration of nanoparticles, followed by measurement of effect on leukocyte proliferation, such as by 3H-thymidine incorporation.	Quantitative analysis of lymphocyte activation and proliferation.	Some assays require radioisotopes, which are biological hazards.	[168–170]