4-Nitro blue tetrazolium chloride, crystals

3,3′-(3,3′-Dimethoxy-4,4′-biphenylene)bis[2-(4-nitrophenyl)-5-phenyl-2H-tetrazolium chloride], 2,2′-bis(4-Nitrophenyl)-5,5′-diphenyl-3,3′-(3,3′-dimethoxy-4,4′-diphenylene)ditetrazolium chloride, p-Nitrotetrazolium blue, Nitro BT, NBT, Nitrotetrazolium Blue chloride, p-Nitro-Blue tetrazolium chloride
Linear Formula:
C40H30N10O6 · 2Cl
CAS Number:
Molecular Weight:
Beilstein/REAXYS Number:
MDL number:
PubChem Substance ID:
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pkg of 5 g

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General description

NBT is soluble in water, aqueous buffers, and dimethylformamide.
Tetrazolium salts are useful in determining the metabolic activity of cells from many origins. The quaternary tetrazole ring core containing four nitrogen atoms, with a net positive charge induces cellular uptake through plasma membrane potential. This drives its use in cell biology. In immunolabeling technique, 4-Nitro blue tetrazolium (NBT) serves as an oxidant. The hydrolysis of 5-bromo-4-chloro-3-indolyl phosphate (BCIP) by alkaline phosphatase gives rise to leucoindigo, which is then being reduced by NBT to form an insoluble blue BCI. Certain cells reduce NBT, which is a colorless/yellow dye to blue or black formazan crystals. Due to this property, NBT is prefered in redox histochemistry and in biochemical applications. It is formerly used to distinguish between nonbacterial and bacterial diseases, the latter causing neutrophils to reduce the dye; used to confirm diagnosis of chronic granulomatous disease.


  • Colony and plaque hybridization
  • Immunohistocytochemistry
  • In situ hybridization
  • Northern blot
  • Southern blot
  • Western blot
NBT has been used in in situ hybridization.


Performance-controlled in the LDH assay.

Preparation Note

Working solution: Solubility: soluble in water, aqueous buffers, and dimethylformamide. Can be dissolved in methanol. A clear solution is achieved at 20 mg/ml.

Analysis Note

Absorption: NBT has its maximum absorption at 260 nm [in water],
and the final product formazan has its maximum absorption at 605 nm [in nitrobenzole].

Other Notes

For life science research only. Not for use in diagnostic procedures.


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Hazard Statements

Target Organs

Respiratory system


NONH for all modes of transport

Flash Point(F)

does not flash

Flash Point(C)

does not flash

Certificate of Analysis
Certificate of Origin
Tetrazolium dyes as tools in cell biology: new insights into their cellular reduction.
Berridge M V, et al.
Biotechnology Annual Review, 11, 127-152 (2005)
Infection and nitroblue-tetrazolium reduction by neutrophils: A diagnostic aid.
Park B H, et al.
Lancet, 292(7567), 532-534 (1968)
D Liebhart et al.
Journal of comparative pathology, 135(4), 237-242 (2006-10-31)
For use in an in-situ hybridization method, three probes (HM, TR and BL) were designed to hybridize, respectively, with (1) Histomonas meleagridis, (2) Tetratrichomonas gallinarum, and (3) a broad range of micro-organisms, including Blastocystis spp. Mono-eukaryotic cultures were used to...
Fluorescent in situ hybridization employing the conventional NBT/BCIP chromogenic stain.
Trinh L A, et al.
Biotechniques, 42(6), 756-759 (2007)
Marina Zieger et al.
PloS one, 9(9), e106562-e106562 (2014-09-06)
We aimed to investigate fractalkine (CX3CL1) protein expression in wild type (wt) retina and its alterations during retinal degeneration in mouse model (rd10) of retinitis pigmentosa. Forms of retinal protein CX3CL1, total protein and mRNA levels of CX3CL1 were analyzed...

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