- Macrophage inducible C-type lectin (Mincle), which signals through Fc receptor gamma chain (FcRγ), spleen tyrosine kinase (Syk), and the adaptor protein Card9, leading to NF-κB activation. [1]
- Nlrp3 inflammasome, leading to caspase-1 activation and IL-1β secretion. [1]

- TDB (5–300 μg/mL) induced dose-dependent IL-1β secretion in LPS-primed bone marrow-derived dendritic cells (BMDCs) from wild-type mice, comparable to the natural cord factor TDM and other particulate inflammasome activators like MSU. [1]
- TDB induced caspase-1 activation (p10 subunit detected in supernatant by Western blot). The pan-caspase inhibitor z-VAD-FMK strongly reduced IL-1β secretion but did not affect TNF-α production. [1]
- IL-1β secretion in response to TDB was abolished in BMDCs from Nlrp3⁻/⁻, Asc⁻/⁻, and caspase-1⁻/⁻ mice, while TNF-α production was unaffected or increased. [1]
- Card9⁻/⁻ BMDCs showed reduced IL-1β and TNF-α secretion when unprimed, but LPS priming restored IL-1β secretion to wild-type levels, indicating Card9 is required for priming but not for inflammasome activation. [1]
- Phagocytosis inhibition with cytochalasin D (100 nM), lysosomal acidification inhibition with bafilomycin A1 (100–400 nM), and cathepsin inhibition with CA-074-Me (10 μM) all significantly reduced TDB-induced IL-1β secretion, but did not affect ATP-induced IL-1β. [1]
- Potassium efflux inhibition with glibenclamide (50 μM) or high extracellular KCl (50 mM) reduced TDB-induced IL-1β secretion and caspase-1 activation. ROS scavengers APDC (500 μM) and ebselen (2 μM) similarly reduced IL-1β secretion without affecting TNF-α. [1]
- Chemokine production (MCP-1, MIP-1α, MIP-1β, RANTES, KC, MIP-2) in response to TDB was unaffected by Nlrp3 deficiency. [1]
- In DDA liposomes (dimethyldioctadecylammonium), incorporation of TDB (6.4–20 mol%) significantly improved colloidal stability, preventing particle size increase during storage at 4°C and 25°C. DDA liposomes without TDB aggregated rapidly. [2]
- DSC analysis showed that TDB incorporated into DDA bilayers, causing a concentration-dependent decrease in phase transition temperature (Tm from 47.2°C to 41.8°C) and broadening of the transition peak, indicating lateral phase separation. [2]
- DDA-TDB liposomes adsorbed 67–79% of the Ag85B-ESAT-6 antigen (negatively charged, pI 4.8) via electrostatic interaction. Antigen retention was ~80% after 7 days at 4°C and remained stable for DDA-TDB, while DDA alone continued to lose antigen. [2]
- Cryo-TEM and TEM confirmed that DDA-TDB liposomes (11 mol% TDB) formed unilamellar vesicles, predominantly below 500 nm, with less aggregation than DDA alone. [2]

- Intraperitoneal injection of TDB (100 μg in 200 μL PBS) into wild-type mice induced significant neutrophil recruitment (CD11b⁺Ly-6G⁺) to the peritoneal cavity at 6 h. This recruitment was significantly reduced in Nlrp3⁻/⁻ mice. [1]
- Subcutaneous immunization of C57BL/6 mice with Ag85B-ESAT-6 (2 μg/dose) in DDA-TDB liposomes (11 mol% TDB, 250 μg DDA, 50 μg TDB per dose) three times at 2-week intervals induced a strong Th1-type immune response: high IFN-γ production from blood lymphocytes upon antigen restimulation, and high IgG2b antibody titers (IgG2b/IgG1 ratio ~0.2). DDA alone induced lower IFN-γ and lower IgG2b. [2]
- CD4⁺ T cells were identified as the primary IFN-γ-producing subset by antibody blocking experiments. [2]
- Compared to alum, DDA-TDB induced significantly higher IFN-γ and IgG2b levels, while alum induced higher IL-5 and IgG1, indicating a Th2 bias. [2]

- ELISA for IL-1β and TNF-α: Cell-free supernatants were collected and cytokine levels were measured by ELISA according to the manufacturer's instructions. [1]
- Western blot for caspase-1 p10: Supernatants were mixed with methanol/chloroform, centrifuged, and the protein pellet was dried, resuspended in sample buffer, separated by NuPAGE, transferred to membranes, and probed with rabbit anti-mouse caspase-1 p10 antibody. [1]
- DSC (Differential Scanning Calorimetry): DDA liposomes with 0–20 mol% TDB were scanned at 30°C/h. Thermodynamic parameters (Tm, ΔHm, ΔT1/2) were calculated. [2]
- Dynamic Light Scattering (DLS): Particle size (z-average diameter) and zeta potential were measured at 25°C. [2]
- Antigen adsorption and retention: 125I-labeled Ag85B-ESAT-6 was added to liposomes, ultracentrifuged (100,000 × g, 1 h), and radioactivity in pellets was measured. [2]

- BMDC generation and stimulation: Bone marrow cells from mice were differentiated with GM-CSF (20 ng/mL) for 7 days. BMDCs were primed with LPS (50 ng/mL, 2 h) and then stimulated with TDB (5–300 μg/mL), TDM, alum (500 μg/mL), MSU (50 μg/mL), or ATP (5 mM). Inhibitors were added 1 h before stimulation. Supernatants were collected for cytokine measurement. [1]
- Lymphocyte cultures: Blood or splenocytes from immunized mice were cultured with Ag85B-ESAT-6 (5 μg/mL) for 72 h. IFN-γ and IL-5 were measured by ELISA. For subset blocking, anti-CD4 or anti-CD8 antibodies were added 30 min before antigen restimulation. [2]

- In vivo peritonitis model: Wild-type and Nlrp3⁻/⁻ mice were injected intraperitoneally with TDB (100 μg in 200 μL PBS) or PBS alone. After 6 h, peritoneal cavities were washed, and neutrophils (CD11b⁺Ly-6G⁺) were quantified by FACS. [1]
- Immunization protocol: Female C57BL/6 mice (8–12 weeks old) were immunized subcutaneously at the base of the tail with 0.2 mL of vaccine containing Ag85B-ESAT-6 (2 μg/dose) in DDA-TDB liposomes (DDA 250 μg, TDB 25–100 μg per dose) or in alum (500 μg). Immunizations were given three times with 2-week intervals. Blood was collected 7 days after the last immunization. [2]

- TDB is described as a less toxic synthetic analogue of trehalose-6,6'-dimycolate (TDM), with shorter fatty acid chains (22 carbons, behenic acid) associated with lower toxicity. [2]
- No body weight loss or overt toxicity was reported in mice immunized with DDA-TDB liposomes. [2]

[1]. The mycobacterial cord factor adjuvant analogue trehalose-6,6'-dibehenate (TDB) activates the Nlrp3 inflammasome. Immunobiology. 2013 Apr;218(4):664-73.
[2]. Characterization of cationic liposomes based on dimethyldioctadecylammonium and synthetic cord factor from M. tuberculosis (trehalose 6,6'-dibehenate)-a novel adjuvant inducing both strong CMI and antibody responses. Biochim Biophys Acta. 2005 Dec 10;1718(1-2):22-31.

Trehalose-6,6'-dibenzene is a fatty acid derivative and also an O-acyl carbohydrate.
- TDB is a synthetic analogue of the mycobacterial cord factor trehalose-6,6'-dimycolate (TDM), a potent pro-inflammatory PAMP. It is insoluble in water and has a particulate appearance in cell culture. [1]
- TDB is used as an adjuvant in combination with the cationic surfactant DDA (dimethyldioctadecylammonium) in tuberculosis subunit vaccines. The DDA-TDB adjuvant system induces both strong cell-mediated immunity (Th1-type) and antibody responses. [2]
- DDA-TDB liposomes (11 mol% TDB) are physically stable and effectively adsorb the negatively charged antigen Ag85B-ESAT-6 (pI 4.8) via electrostatic interactions. [2]
- The mechanism of action of TDB involves two parallel pathways: (1) Mincle-FcRγ-Syk-Card9 signaling leading to NF-κB activation and pro-IL-1β production; (2) Nlrp3 inflammasome activation leading to caspase-1-dependent IL-1β maturation and secretion. [1]

C56H106O13

987.433040000001

986.763

C, 68.12; H, 10.82; O, 21.06

66758-35-8

11170611

White to off-white solid powder

11.348

6

13

48

69

1100

10

CCCCCCCCCCCCCCCCCCCCCC(=O)OC[C@@H]1[C@H]([C@@H]([C@H]([C@H](O1)O[C@@H]2[C@@H]([C@H]([C@@H]([C@@H](COC(=O)CCCCCCCCCCCCCCCCCCCCC)O2)O)O)O)O)O)O

ZLJJDBSDZSZVTF-LXOQPCSCSA-N

InChI=1S/C56H106O13/c1-3-5-7-9-11-13-15-17-19-21-23-25-27-29-31-33-35-37-39-41-47(57)65-43-45-49(59)51(61)53(63)55(67-45)69-56-54(64)52(62)50(60)46(68-56)44-66-48(58)42-40-38-36-34-32-30-28-26-24-22-20-18-16-14-12-10-8-6-4-2/h45-46,49-56,59-64H,3-44H2,1-2H3/t45-,46-,49-,50-,51+,52+,53-,54-,55-,56-/m1/s1

[(2R,3S,4S,5R,6R)-6-[(2R,3R,4S,5S,6R)-6-(docosanoyloxymethyl)-3,4,5-trihydroxyoxan-2-yl]oxy-3,4,5-trihydroxyoxan-2-yl]methyl docosanoate

Trehalose-6,6'-dibehenate; 22:0 Trehalose; D-(+)-trehalose 6,6'-dibehenate; Trehalose 6,6'-dibehenate; 672NB7JH7R; DTXSID30457482;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)