Tetrazolium Red

Tetrazolium Red (2,3,5-Triphenyltetrazolium chloride; TTC) is a not brain-penetrant, colorless, water-soluble dye that is reduced by mitochondrial enzymes to a deep red, water-insoluble compound (formazan) mainly in the mitochondria of living cells. Tetrazolium Red is used to observe the activity of dehydrogenase, and it turns colorless to red when exposed to hydrogen. Tetrazolium Red distinguishes between surviving and infarcted brain tissue after stroke. Tetrazolium Red has been used to stain heart tissue to measure the extent of acute lesions and also used to stain brain tissue to detect the size of the infarcted area. The absorption wavelength of Tetrazolium Red is 570 nm^[1][2][3][4]. In Vitro:Tetrazolium Red staining of the size of the ischemic area^[3]:
1) Preparation of human umbilical mesenchymal stem cells (HUMSCs)
HUMSCs are dissociated from the infants and cultured by 10% fetal bovine serum in DMEM at 37℃.
2) Animal Surgery
The rats are are anesthetized with chloral hydrate (400 mg/kg, i.p.) and are performed MCAO surgery and reperfusion.
NOTE: MCAO surgery, a hole was drilled by the right orbit to expose the right middle cerebral artery. The artery was ligated at the same time as both common carotid arteries were clamped simultaneously for 90 minutes, after which the ligature and clamping clips were removed to restore blood flow.
3) Transplantation of HUMSCs
A total of 5x10⁵ HUMSCs are grafted into the infarct cortex of each rat 24 hours after MCAO by 2 injections.
4) Infarct Cortex Identification
Rats are deeply anesthetized and decapitated. Coronal sections of the brains were sliced at 2 mm, immersed in 2% Tetrazolium Red, and then fixed with 10% formalin.
5) Data analysis (digital images)
The size of the infarct area, which is devoid of red staining is determined on the digital images using ImagePro software.
Tetrazolium Red staining of cell proliferation and viability measurement^[4]:
1) Preparation: 2-5 mg/mL Tetrazolium Red in PBS.
2) Isolated cells are counted manually with a hemocytometer and then various concentrations (5x10⁴ to 1x10⁶ are plated into 24-well culture plates, fed 1 mL of control culture media and returned to the incubator for a 24 h period to allow for cell to reattach to the plastic culture plates.
3) The next day, media is removed and cells are fed 1 mL fresh media, and then 200 mL of 5 mg/mL Tetrazolium Red is added to each well so that final concentration is 0.83 mg/mL Tetrazolium Red. Plates are returned to the incubator (37℃) for a 4 h incubation period.
4) Media is removed and 1 mL of isopropanol is added to each well to dissolve the formazan crystals. Optical density is measured at 570 nm.
In Vivo:Tetrazolium Red staining of brain damage and the size of the infarct area^[1]：
1)For lipopolysaccharide (LPS)-sensititized neonatal cerebral hypoxia-ischemia (HI), LPS (0.3 mg/kg) is i.p.-injected to 5-day-old CD1 mice at 72 h before HI. At postnatal day 8, mice are anesthetized by 2% isoflurane and subjected to permanent ligation of the right common carotid artery. Mice recover for 1.5 h and are then exposed to hypoxia in glass chambers containing 10% oxygen and 90% N2 in a waterbacth kept at 37℃. After hypoxic exposure, mice are returned to dams in the animal care facility. The in-vivo Tetrazolium Red-labeling procedure is performed at 48 h recovery.
2)Mannitol (0.5 M-1.4M) prepared in PBS at a temperature of 37℃ is IP-injected to animals (~0.1 mL/g body weight) for 5 to 180 min to disrupt blood-brain-barrier (BBB). Mice are anesthetized with avertin and transcardially perfused of PBS followed by 10 mL of 2% Tetrazolium Red. At 10 min after transcardial Tetrazolium Red perfusion, the brains of animals are removed and placed into 4% paraformaldehyde. Alternatively, the extracted brains can be incubated in warm phosphate-saline buffer (PBS) for 30 min to enhance Tetrazolium Red-staining. Brains are removed and divided into the contralateral (the left cortex) and lesion sides (the right cortex) for protein extraction.
3)Data analysis (digital images): Brain damage is expressed as the ratio of the infarcted area (white area) to the area of the undamged, contralateral hemisphere.