Cat. No.: HY-14536

Methylene Blue

(Synonyms: Basic Blue 9; CI-52015; Methylthioninium chloride)

30 Cited Publications

Customer Review

Based on 30 publication(s) in Google Scholar

Methylene blue (Basic Blue 9) is a guanylyl cyclase (sGC), monoamine oxidase A (MAO-A) and NO synthase (NOS) inhibitor. Methylene blue is a vasopressor and is often used as a dye in several medical procedures. Methylene blue through the nitric oxide syntase/guanylate cyclase signalling pathway to reduce prepulse inhibition. Methylene blue is a REDOX cycling compound and able to cross the blood-brain barrier. Methylene blue is a Tau aggregation inhibitor. Methylene Blue is a photosensitizer and redox agent. Methylene blue reduces cerebral edema, attenuated microglial activation and reduced neuroinflammation.

For research use only. We do not sell to patients.

Purity: 95.05%
CAS No.: 61-73-4
Formula: C₁₆H₁₈ClN₃S
Molecular Weight:319.85

Storage:

RT, sealed storage, away from moisture and light.

In solvent -80°C, 1 year , -20°C, 6 months

Methylene Blue Chemical Structure

CAS No.: 61-73-4

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Top Publications Citing

Use of MCE Products

Fluorescent Dye

Strong Fluorescence Signal / Multi-Disciplinary Applications / High Fluorescence Specificity

Biological Activity
- Description
- IC₅₀ & Target
- Cellular Effect
- In Vitro
- In Vivo
- Dyeing Example
Chemical Information
Publications (30)
Solvent & Solubility
- In Vitro
Purity & Documentation
References
Help & FAQs
Classifications
Bioz

Biological Activity

Description

IC₅₀ & Target

Guanylyl cyclase (sGC)^[1].
Monoamine oxidase A (MAO-A)^[1].
NO synthase (NOS)^[1]

All Monoamine Oxidase Isoforms :

MAO-A
MAO-B

All NO Synthase Isoforms :

Cellular Effect

Cell Line	Type	Value	Description	References
B16-F10	IC₅₀	5.48 μM Compound: MB	Antiproliferative activity against mouse B16-F10 cells assessed as cell viability in light irradiation and measured for 48 hrs by MTT assay Antiproliferative activity against mouse B16-F10 cells assessed as cell viability in light irradiation and measured for 48 hrs by MTT assay	[PMID: 34871840]
B16-F10	IC₅₀	7.02 μM Compound: MB	Antiproliferative activity against mouse B16-F10 cells assessed as cell viability in dark incubated for 48 hrs by MTT assay Antiproliferative activity against mouse B16-F10 cells assessed as cell viability in dark incubated for 48 hrs by MTT assay	[PMID: 34871840]
Fibroblast	EC₅₀	43 nM Compound: MB	Inhibition of erastin-induced cell death in FRDA patient-derived fibroblast assessed as depletion of cellular ATP incubated for 12 hrs followed by erastin stimulation and measured after by luciferase-linked ATPase enzymatic assay Inhibition of erastin-induced cell death in FRDA patient-derived fibroblast assessed as depletion of cellular ATP incubated for 12 hrs followed by erastin stimulation and measured after by luciferase-linked ATPase enzymatic assay	[PMID: 33214825]
FM3A	EC₅₀	1.1 x 10^-6M Compound: Methylene blue	Cytotoxicity against mouse mammary tumor FM3A cells representing a model of host Cytotoxicity against mouse mammary tumor FM3A cells representing a model of host	[PMID: 11855978]
HepG2	IC₅₀	<1 μM Compound: Methylene blue	Antimalarial activity against sporozoite stage of Plasmodium yoelii assessed as invasion of human HepG2 cells expressing CD81 incubated for 2 hrs prior to inoculation measured after 1 hr by immunofluorescence assay in presence of penicillin/streptomycin Antimalarial activity against sporozoite stage of Plasmodium yoelii assessed as invasion of human HepG2 cells expressing CD81 incubated for 2 hrs prior to inoculation measured after 1 hr by immunofluorescence assay in presence of penicillin/streptomycin	[PMID: 23927658]
HepG2	IC₅₀	6 μM Compound: MB	Anticancer activity against human HepG2 cells Anticancer activity against human HepG2 cells	[PMID: 33460833]
HT-29	IC₅₀	>10 μM Compound: MB	Cytotoxicity against human HT-29 cells assessed as reduction in cell viability incubated for 24 hrs in dark by MTT assay Cytotoxicity against human HT-29 cells assessed as reduction in cell viability incubated for 24 hrs in dark by MTT assay	[PMID: 32267685]
HT-29	IC₅₀	9.2 μM Compound: MB	Cytotoxicity against human HT-29 cells assessed as reduction in cell viability preincubated for 1 hr followed by light irradiation at 3.2 J/cm2 for 30 sec and measured after 24 hrs by MTT assay Cytotoxicity against human HT-29 cells assessed as reduction in cell viability preincubated for 1 hr followed by light irradiation at 3.2 J/cm2 for 30 sec and measured after 24 hrs by MTT assay	[PMID: 32267685]
Lymphocyte	EC₅₀	88 nM Compound: MB	Anti-ferroptotic activity in FRDA patient-derived Lymphocyte assessed as reduction in RSL3-induced lipid peroxidation incubated for overnight followed by RSL3 stimulation and measured after 90 mins by FACS analysis Anti-ferroptotic activity in FRDA patient-derived Lymphocyte assessed as reduction in RSL3-induced lipid peroxidation incubated for overnight followed by RSL3 stimulation and measured after 90 mins by FACS analysis	[PMID: 33214825]
MCF7	IC₅₀	10.58 μM Compound: MB	Antiproliferative activity against human MCF7 cells assessed as cell viability in light irradiation and measured after 48 hrs by MTT assay Antiproliferative activity against human MCF7 cells assessed as cell viability in light irradiation and measured after 48 hrs by MTT assay	[PMID: 34871840]
MCF7	IC₅₀	14.64 μM Compound: MB	Antiproliferative activity against human MCF7 cells assessed as cell viability in dark incubated for 48 hrs by MTT assay Antiproliferative activity against human MCF7 cells assessed as cell viability in dark incubated for 48 hrs by MTT assay	[PMID: 34871840]

In Vitro

Methylene blue (Basic Blue 9) (4.5 μM; BV2 microglia) alters the immune profile of LPS-activated BV2 microglia and decreases the level of CD14, IL-1β, TNF-α, and CCL2 mRNA^[3].
1. Sample treatment:
1.1 For paraffin sections: dewax and rehydrate as usual.
1.2 For frozen sections: distilled water for 2 min.
1.3 For cultured cells: Fix with 4% paraformaldehyde. Wash with distilled water for 2 min. Replace with fresh distilled water and wash for another 2 min.
2. Methylene blue staining
2.1 Stain with 0.1% Methylene blue for 2 min.
2.2 Wash thoroughly with distilled water, observe and take photos.
2.3 Staining results:
Tissue or cells: blue

MedChemExpress (MCE) has not independently confirmed the accuracy of these methods. They are for reference only.

In Vivo

Methylene blue (Basic Blue 9) (50 and 100 mg/kg; i.p.; once, for 25 min; male NMRI mice) reduces absent prepulse inhibition^[1].
Methylene blue (Basic Blue 9) (20 and 40 mg/kg; p.o.; daily, for 6 months; CaMKIIα-tTA transactivator mice) preserves cognition in mice expressing full-length pro-aggregant human Tau^[2].
Methylene blue (Basic Blue 9) (2 mg/kg; i.v.; once, for 1 d; TBI-treated male BALB/c mice) reduces TBI-induced edema and neuroinflammation and reduces acute depression-like behavior^[3].
Methylene blue (Basic Blue 9) (2 mg/kg; i.v.; once, for 1 d; TBI-treated male BALB/c mice) reduces the percentage of inflammatory factor^[3].

MedChemExpress (MCE) has not independently confirmed the accuracy of these methods. They are for reference only.

Animal Model:

Male NMRI mice^[1]
Dosage:

50 and 100 mg/kg
Administration:

Intraperitoneal injection; once, for 25 minutes
Result:

Reduced the prepulse inhibition and reduced the increase in locomotor activity caused by phencyclidine (PCP).

Animal Model:

CaMKIIα-tTA transactivator mice^[2]
Dosage:

20 and 40 mg/kg
Administration:

Oral administration; daily, for 6 months
Result:

Inhibited Tau aggregation in CaMKIIα-tTA transactivator mice.

Animal Model:

TBI-treated male BALB/c mice^[3]
Dosage:

2 mg/kg
Administration:

Intravenous injection; once, for 1 day
Result:

Decreased the level of CD14, IL-1β, TNF-α, and CCL2 mRNA.

Animal Model:

TBI-treated male BALB/c mice^[3]
Dosage:

2 mg/kg
Administration:

Intravenous injection; once, for 1 day
Result:

Reduced the percentage of myeloid (CD11b+/GR1+) cells, reduced IL-1β and enhanced IL-10 expression in microglia.

Dyeing Example

Chemical Information

CAS No. 61-73-4
Appearance Solid
Molecular Weight 319.85
Formula C₁₆H₁₈ClN₃S
Color Brown to reddish brown
SMILES

CN(C)C1=CC=C2C(SC(C(C=C/3)=N2)=CC3=[N+](C)/C)=C1.[Cl-]
Synonyms

Basic Blue 9; CI-52015; Methylthioninium chloride
Shipping

Room temperature in continental US; may vary elsewhere.

Storage

RT, sealed storage, away from moisture and light

In solvent	-80°C	1 year
	-20°C	6 months

Publications (30)

All Gel ElectrophoresisCell Migration/Invasion AssayWB

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Journal Impact Factor

ACS Nano

Keratinocyte-Derived Apoptotic Nanovehicles Reprogram Cutaneous Dendritic Cells to Ameliorate Psoriasis. [Abstract]

2026 Jun 30;20(25):18058-18072. PMID: 42301004
Nat Commun

DEAD-box RNA helicase 10 is required for 18S rRNA maturation by controlling the release of U3 snoRNA from pre-rRNA in embryonic stem cells. [Abstract]

2024 Nov 27;15(1):10303. PMID: 39604362

Methylene Blue purchased from MedChemExpress. Usage Cited in: Nat Commun. 2024 Nov 27;15(1):10303. [Abstract]

Northern blot analysis of pre-rRNA intermediates using 5'ETS-1, 5'ETS-2, 5'ETS and ITS1-29 probes in DDX10-AID mESCs with IAA treatment (0 h, 6 h, 24 h, and 48 h) or treated with IAA for 48 h followed by 48 h of washing. MB represents methylene blue staining. Experiments were repeated three times independently with similar results. Membranes were stained with Methylene Blue stain solution (0.03% Methylene Blue in 0.3 M sodium acetate (pH 5.0-5.5)), and then RNA was crosslinked onto the membrane using UV light.

Methylene Blue purchased from MedChemExpress. Usage Cited in: Nat Commun. 2024 Nov 27;15(1):10303. [Abstract]

Northern blot analysis of pre-rRNA intermediate levels in DDX10-AID (+ OsTir1) mESCs overexpressing DDX10FL, DDX10ΔHBD, DDX10ΔHCD or DDX10ΔHD at 0 h and 48 h after IAA treatment. MB represents Methylene Blue staining. Experiments were repeated three times independently with similar results. Source data are provided as a Source Data file. Membranes were stained with Methylene Blue stain solution (0.03% Methylene Blue in 0.3 M sodium acetate (pH 5.0-5.5)), and then RNA was crosslinked onto the membrane using UV light.

Methylene Blue purchased from MedChemExpress. Usage Cited in: Nat Commun. 2024 Nov 27;15(1):10303. [Abstract]

Northern blot analysis of pre-rRNA intermediates in DDX10-AID (+ OsTir1) mESCs overexpressing NUP98-DDX10 or DDX10, treated with IAA at different time points (0 h, 24 h, and 48 h). MB represents Methylene Blue staining. Experiments were repeated two times independently with similar results. Source data are provided as a Source Data file. Membranes were stained with Methylene Blue stain solution (0.03% Methylene Blue in 0.3 M sodium acetate (pH 5.0-5.5)), and then RNA was crosslinked onto the membrane using UV light.
Theranostics

Establishment of tumor inflammasome clusters with distinct immunogenomic landscape aids immunotherapy. [Abstract]

2021 Oct 17;11(20):9884-9903. PMID: 34815793

Methylene Blue purchased from MedChemExpress. Usage Cited in: Theranostics. 2021 Oct 17;11(20):9884-9903. [Abstract]

Migration analysis of THP-1, co-cultured with cancer cells that were pre-treated with Methylene Blue.

Methylene Blue purchased from MedChemExpress. Usage Cited in: Theranostics. 2021 Oct 17;11(20):9884-9903. [Abstract]

Western blotting analysis of PD-L1 expression in THP-1 which was co-cultured with cancer cells pre-treated with Methylene Blue.
Chem Eng J

2025 Nov 24;526:171230.
Biomaterials

Manganese-enriched photonic/catalytic nanomedicine augments synergistic anti-TNBC photothermal/nanocatalytic/immuno-therapy via activating cGAS-STING pathway. [Abstract]

2023 Feb:293:121988. PMID: 36580716
J Nanobiotechnology

SIRT5-modified human umbilical cord mesenchymal stem cells loaded with antioxidant polydopamine nanozyme enhance parpi resistance in ovarian cancer via fatty acid metabolism reprogramming. [Abstract]

2025 Jul 4;23(1):485. PMID: 40616128
Redox Biol

An effective inactivant based on singlet oxygen-mediated lipid oxidation implicates a new paradigm for broad-spectrum antivirals. [Abstract]

2020 Sep;36:101601. PMID: 32535542
Gut Microbes

Helicobacter pylori infection aggravates hepatic steatosis by lactylation-driven WTAP-mediated m6A modification. [Abstract]

2025 Dec 31;17(1):2599543. PMID: 41384837
Nano Res

2026 Feb 6.
Phytomedicine

Arnicolide C induces ROS-mediated modulation of PI3K/Akt and MAPK pathways to suppress MYC in hepatocellular carcinoma. [Abstract]

2025 Oct 20:148:157423. PMID: 41138574
Cell Rep

WDFY2 promotes MRN complex formation required for homologous recombination-mediated DNA repair. [Abstract]

2025 Nov 25;44(11):116520. PMID: 41196680
Cell Rep

The ARID1A-METTL3-m6A axis ensures effective RNase H1-mediated resolution of R-loops and genome stability. [Abstract]

2024 Feb 13;43(2):113779. PMID: 38358891
Nanophotonics

Disentangling the absorption lineshape of methylene blue for nanocavity strong coupling. [Abstract]

2025 Nov 25;14(27):4993-5001. PMID: 41426083
J Environ Sci

2025 Dec 25.
Phytother Res

Carnosol inhibits KGN cells oxidative stress and apoptosis and attenuates polycystic ovary syndrome phenotypes in mice through Keap1-mediated Nrf2/HO-1 activation. [Abstract]

2023 Apr;37(4):1405-1421. PMID: 36786429
Chemistry

2026 May;8(5):65.
Bioengineering (Basel)

LIPUS Enhances Gallbladder Motility via ANO1 in Acute Cholecystitis Guinea Pigs. [Abstract]

2025 Oct 27;12(11):1164. PMID: 41301120
Cell Signal

YTHDF2 drives oral squamous cell carcinoma progression via m6A-dependent degradation of MTUS1/ATIP1 mRNA and mitochondrial dysregulation. [Abstract]

2025 Sep 18:136:112145. PMID: 40975502
BMC Cancer

FTO-mediated demethylation of MTUS1/ATIP1 promotes tumor progression in head and neck squamous cell carcinoma. [Abstract]

2024 Dec 3;24(1):1489. PMID: 39627705
Biomed Res Int

Gentiopicroside Produces Endothelium-Independent Vasodilation by Deactivating the PI3K/Akt/Rho-Kinase Pathway in Isolated Rat Thoracic Aorta. [Abstract]

2021 May 14:2021:5565748. PMID: 34095301
Exp Ther Med

MicroRNA-139-5p regulates chronic inflammation by suppressing nuclear factor-κB activity to inhibit cell proliferation and invasion in colorectal cancer. [Abstract]

2019 Nov;18(5):4049-4057. PMID: 31616518
World J Gastrointest Surg

Impact of interstitial cells of Cajal on slow wave and gallbladder contractility in a guinea pig model of acute cholecystitis. [Abstract]

2023 Jun 27;15(6):1068-1079. PMID: 37405098
Hematology

Downregulation of Smad4 expression confers chemoresistance against imatinib mesylate to chronic myeloid leukemia K562 cells. [Abstract]

2022 Dec;27(1):43-52. PMID: 34957936
Res Sq

2025 Nov 12.
Patent

US20240325538A1.
Patent

US20240238271A1.
Patent

US20230226111A1.
Research Square Print

2023 Feb 21.
Curr Res Virol Sci

Identification of broad anti-coronavirus chemical agents for repurposing against SARS-CoV-2 and variants of concern. [Abstract]

2022;3:100019. PMID: 35072124
Evid Based Complement Alternat Med

Acupuncture at Gastric Back-Shu and Front-Mu Acupoints Enhances Gastric Motility via the Inhibition of the Glutamatergic System in the Hippocampus. [Abstract]

2020 Mar 10;2020:3524641. PMID: 32215036

Solvent & Solubility

In Vitro:

H₂O : 50 mg/mL (156.32 mM; Need ultrasonic)

View the Complete Stock Solution Preparation Table

Preparing Stock Solutions

View the Complete Stock Solution Preparation Table

Concentration Solvent Mass

1 mg

5 mg

10 mg

Concentration

Solvent

Mass

1 mM

3.1265 mL

15.6323 mL

31.2647 mL

5 mM

0.6253 mL

3.1265 mL

6.2529 mL

10 mM

0.3126 mL

1.5632 mL

3.1265 mL

Please refer to the solubility information to select the appropriate solvent. Once prepared, please aliquot and store the solution to prevent product inactivation from repeated freeze-thaw cycles.

Storage method and period of stock solution: -80°C, 1 year; -20°C, 6 months. When stored at -80°C, please use it within 1 year. When stored at -20°C, please use it within 6 months.

* Note: If you choose water as the stock solution, please dilute it to the working solution, then filter and sterilize it with a 0.22 μm filter before use.

Molarity Calculator

Mass (g) = Concentration (mol/L) × Volume (L) × Molecular Weight (g/mol)

Mass Mass

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Volume Volume

Molecular Weight Molecular Weight

Dilution Calculator

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Purity & Documentation

Select Batch:

Purity: 99.98%

Assay: 99.98%

ee.: 99.98%

Data Sheet (279 KB)
SDS (393 KB)
- English - EN (393 KB)
- Français - FR (393 KB)
- Deutsch - DE (393 KB)
- Norwegian - NO (393 KB)
- Español - ES (393 KB)
- Swedish - SV (393 KB)
- Italian - IT (393 KB)
- Korean - KR (393 KB)
- Portuguese - PT (393 KB)
COA (2659 KB)
Elemental Analysis Report (2659 KB)
HNMR (2659 KB)
CNMR (2659 KB)
RP-HPLC (2659 KB)
NP-HPLC (2659 KB)
MS (2659 KB)
LCMS (2659 KB)
IR (2659 KB)
GC (2659 KB)
OR (2659 KB)
SDS-PAGE (2659 KB)
SEC-HPLC (2659 KB)
Other Report (2659 KB)
Handling Instructions (2659 KB)

References

[1]. Klamer D, et, al. Phencyclidine-induced behaviour in mice prevented by methylene blue. Basic Clin Pharmacol Toxicol. 2004 Feb;94(2):65-72. [Content Brief]

[2]. Hochgräfe K, et, al. Preventive methylene blue treatment preserves cognition in mice expressing full-length pro-aggregant human Tau. Acta Neuropathol Commun. 2015 May 10;3:25. [Content Brief]

[3]. Fenn AM, et, al. Methylene blue attenuates traumatic brain injury-associated neuroinflammation and acute depressive-like behavior in mice. J Neurotrauma. 2015 Jan 15;32(2):127-38. [Content Brief]

[4]. Kalaitzakis D, et al. Methylene Blue as a Photosensitizer and Redox Agent: Synthesis of 5-Hydroxy-1H-pyrrol-2(5H)-ones from Furans. Angew Chem Int Ed Engl. 2015 May 18;54(21):6283-7. [Content Brief]

Help & FAQs

How should lyophilized recombinant proteins be reconstituted and stored?

1. Before opening the cap, centrifuge the vial at 13000 rpm for 20-30 seconds. This step will ensure that any lyophilized powder that may have adhered to the cap or walls is collected at the bottom of the vial, minimizing the risk of product loss.

Complete Stock Solution Preparation Table

Storage method and period of stock solution: -80°C, 1 year; -20°C, 6 months. When stored at -80°C, please use it within 1 year. When stored at -20°C, please use it within 6 months.

Optional Solvent	Concentration Solvent Mass	1 mg	5 mg	10 mg	25 mg
H₂O	1 mM	3.1265 mL	15.6323 mL	31.2647 mL	78.1616 mL
	5 mM	0.6253 mL	3.1265 mL	6.2529 mL	15.6323 mL
	10 mM	0.3126 mL	1.5632 mL	3.1265 mL	7.8162 mL
	15 mM	0.2084 mL	1.0422 mL	2.0843 mL	5.2108 mL
	20 mM	0.1563 mL	0.7816 mL	1.5632 mL	3.9081 mL
	25 mM	0.1251 mL	0.6253 mL	1.2506 mL	3.1265 mL
	30 mM	0.1042 mL	0.5211 mL	1.0422 mL	2.6054 mL
	40 mM	0.0782 mL	0.3908 mL	0.7816 mL	1.9540 mL
	50 mM	0.0625 mL	0.3126 mL	0.6253 mL	1.5632 mL
	60 mM	0.0521 mL	0.2605 mL	0.5211 mL	1.3027 mL
	80 mM	0.0391 mL	0.1954 mL	0.3908 mL	0.9770 mL
	100 mM	0.0313 mL	0.1563 mL	0.3126 mL	0.7816 mL

* Note: If you choose water as the stock solution, please dilute it to the working solution, then filter and sterilize it with a 0.22 μm filter before use.

Methylene Blue

30 Cited Publications

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Methylene Blue

Publications Citing Use of MedChemExpress (MCE) Methylene Blue

All Monoamine Oxidase Isoforms

All NO Synthase Isoforms

Biological Activity

Chemical Information

Publications (30)

Solvent & Solubility

Purity & Documentation

References

Help & FAQs

Complete Stock Solution Preparation Table

Methylene Blue Related Classifications

HY-14536 Related Classifications