The following figure depicts the mechanisms and subcellular locations of various functional assays commonly used for the charecterization of in vitro oxygen-glucose deprivation model of brain cells. These assays are:(1) DCFDA assay, (2) MTT assay, (3) MPT pore opening assay, (4) LDH assay, (5) FITC Annexin V - PI staining, (6) Trypan blue dye exclusion, (7) Tunnel assay. MTT assay and LDH assay are the two most frequently used techniques.

Reference: Babu M, Singh N, Datta A. (2022). Molecular Neurobiology

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Download image - OGD-Prot: Functional Assay

DCFDA Assay: 2',7'-Dichlorodihydrofluorescein diacetate (DCFDA) assay is used to measure the intracellular ROS. DCFDA is diffused into cells and is deacetylated by cellular esterases to non-fluorescent 2’, 7’-dichlorodihydrofluorescin (DCFH), which is rapidly oxidized to highly fluorescent 2’, 7’-dichlorodihydrofluorescein (DCF) by ROS.(1)

MTT Assay: MTT assay is used to measure cellular metabolic activity as an indicator of cell viability. This colorimetric assay is based on the reduction of a yellow tetrazolium salt (MTT) to purple formazan crystals by metabolically active cells. The viable cells contain NAD(P)H-dependent oxidoreductase enzymes which reduce the MTT to formazan. The insoluble formazan crystals are dissolved using a solubilization solution and the resulting colored solution is quantified by measuring absorbance.(2)

MPT Pore Opening Assay: During cell death, the opening of the mitochondrial permeability transition pore dramatically alters the permeability of mitochondria. MPT pore opening assay measures mitochondrial permeability transition pore opening than assays relying on mitochondrial membrane potential alone. Cells are loaded with the acetoxymethyl ester of calcein dye, calcein AM, which passively diffuses into the cells and accumulates in cytosolic compartments, including the mitochondria. Once inside cells, intracellular esterases cleave the acetoxymethyl esters to liberate the very polar fluorescent dye calcein, which does not cross the mitochondrial or plasma membranes in appreciable amounts over relatively short periods of time. The fluorescence from cytosolic calcein is quenched by the addition of cobalt chloride (CoCl₂), while the fluorescence from the mitochondrial calcein is maintained. As a control, cells that have been loaded with calcein AM and CoCl₂ can also be treated with an ionophore, ionomycin, to allow entry of excess Ca2+ into the cells to trigger mitochondrial pore activation and subsequent loss of mitochondrial calcein fluorescence.(3)

LDH Assay: Lactate dehydrogenase is a cytosolic enzyme present in many different cell types and is a well-defined and reliable indicator of cytotoxicity. Damage to the plasma membrane releases LDH into the surrounding cell culture media. The extracellular LDH in the media can be quantified by a coupled enzymatic reaction in which LDH catalyzes the conversion of lactate to pyruvate via NAD+ reduction to NADH. Oxidation of NADH by diaphorase leads to the reduction of a tetrazolium salt (INT) to a red formazan product that can be quantified by colorimetry.(4)

FITC-Annexin V - Propidium Iodide Staining: Phosphatidylserine is normally confined in the inner membrane leaflet of viable cells. The translocation of phosphatidylserine to the exposed membrane surface is an early event in apoptosis. FITC-Annexin V has a very high affinity for membranes containing the negatively charged phospholipid phosphatidylserine and can be used as a marker of early apoptosis. Propidium Iodide is impermeant to live cells and apoptotic cells, but stains dead cells with red fluorescence when it binds to DNA intercalating between the bases with little or no sequence preference.(5)

Trypan Blue Dye Exclusion: The trypan blue dye exclusion test is used to determine the number of viable cells present in a cell suspension, the live cells possess intact cell membranes that exclude the trypan blue dyes, whereas dead cells do not.(6)

TUNEL Assay: DNA fragmentation in apoptosis is usually associated with structural changes in cellular morphology and is a hallmark of late-stage apoptosis. DNA fragmentation in apoptosis can be examined using the TUNEL assay. The TUNEL assay relies on the terminal deoxynucleotide transferase enzyme to catalyze the addition of labeled dUTP to the 3’-hydroxyl ends of cleaved DNA fragmentations. Labeled dUTP such as biotin-dUTP can be recognized using secondary reagent streptavidin through colorimetric detection.(7)

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