| Size | Price | Stock | Qty |
|---|---|---|---|
| 1mg |
|
||
| Other Sizes |
| Targets |
TLR9 (Toll-like receptor 9). ODN 2216 interacts with TLR9 in the lysosomes of CD4+ T cells and plasmacytoid dendritic cells (pDCs), activating feedback-dependent signaling pathways through the IRAK4/IRF7 axis. This interaction induces robust type I interferon responses, particularly IFN-alpha and IFN-beta, leading to strong Th1-like immune activation and potent CD8+ T-cell responses. As a Class A CpG ODN, ODN 2216 preferentially activates pDCs and induces high levels of IFN-alpha, distinguishing it from other CpG ODN classes that primarily stimulate B cells. The FITC label does not interfere with TLR9 binding specificity.
|
|---|---|
| ln Vitro |
ODN 2216 induces the production of type I interferons (IFN-alpha, IFN-beta), IL-6, and TGF-beta via the IRAK4/IRF7 axis while increasing intracellular ATP levels. It not only induces the differentiation of CD4+ T cells into anti-inflammatory Th3-like regulatory phenotypes to inhibit autologous proliferation but also enhances specific CD8+ T cell-mediated cytotoxicity against target cells. In breast cancer cell models, ODN 2216 enhances CD8+ T cell cytotoxicity against Mammaglobin-A-expressing cancer cells. The FITC-labeled version retains full biological activity, making it suitable for parallel uptake and functional studies.
|
| ln Vivo |
In vivo, ODN 2216 induces strong Th1-like immune activation characterized by robust type I interferon secretion (IFN-alpha and IFN-beta) and activation of CD8+ T cell responses. In murine models of systemic lupus erythematosus, ODN 2216 administration modulates immune responses and influences disease progression. In tumor models, it enhances antigen-specific CD8+ T cell responses, promoting anti-tumor immunity. The compound also induces pDC maturation and activation, leading to downstream cytokine cascades. Plasma cytokine levels can be measured post-administration via ELISA to confirm immune activation. Tissue distribution of FITC-labeled ODN 2216 can be tracked by measuring fluorescence intensity in lymphoid organs after intravenous or subcutaneous injection.
|
| Enzyme Assay |
For non-cellular TLR9 binding assays, a standard ELISA-based binding assay can be employed. Briefly, recombinant human TLR9 protein is immobilized on a 96-well plate overnight at 4degC. After blocking with 1% BSA in PBS, varying concentrations of FITC-labeled ODN 2216 (0-1000 nM) are added and incubated for 2 hours at room temperature. Unbound ODN is removed by washing. Bound FITC-labeled ODN is detected using an anti-FITC-HRP conjugate and TMB substrate, measuring absorbance at 450 nm. Alternatively, SPR can be used: TLR9 is immobilized on a CM5 chip, and FITC-labeled ODN 2216 is flowed over to measure binding affinity (KD). Direct fluorescence-based binding can be assessed by incubating TLR9 with labeled ODN and measuring fluorescence polarization.
|
| Cell Assay |
For cellular uptake and TLR9 activation studies, human PBMCs, plasmacytoid dendritic cells (pDCs), or TLR9-expressing HEK293 reporter cells are seeded in 96-well plates (2-5 × 10^5 cells/well) in RPMI-1640 medium with 10% FBS. Cells are treated with FITC-labeled ODN 2216 at concentrations ranging from 0.1-10 uM for 4-24 hours. Cellular uptake is assessed by confocal laser-scanning microscopy (excitation 495 nm, emission 520 nm) to visualize intracellular localization, or quantified by flow cytometry. Functional activation is measured by collecting supernatants for IFN-alpha, IL-6, and TNF-alpha quantification via ELISA. For TLR9-specificity validation, assays are performed in the presence of the TLR9 antagonist ODN 2088. Live-cell imaging can be performed to track endosomal trafficking dynamics.
|
| Animal Protocol |
For in vivo distribution studies, animals (typically mice) receive a single intravenous (tail vein), intraperitoneal, or subcutaneous injection of FITC-labeled ODN 2216 at doses ranging from 10-50 mg/kg. At various time points post-injection (0.5, 2, 6, 12, 24, 48 hours), blood is collected via cardiac puncture, and lymphoid organs (spleen, lymph nodes), liver, and kidneys are harvested. Tissue homogenates are prepared in lysis buffer, and FITC fluorescence (excitation 485 nm, emission 528 nm) is measured using a fluorescence plate reader. For histological analysis, frozen tissue sections are counterstained with DAPI and examined by fluorescence microscopy to determine cellular localization within lymphoid follicles and other immune compartments. Plasma cytokine levels (IFN-alpha, TNF-alpha, IL-6) are measured by ELISA to correlate uptake with functional immune activation.
|
| ADME/Pharmacokinetics |
Class A CpG ODNs like ODN 2216 exhibit rapid plasma clearance following systemic administration due to their partial phosphodiester backbone, with a plasma half-life of approximately 30-60 minutes in mice. The compound distributes primarily to lymphoid tissues including spleen, lymph nodes, and bone marrow, as well as liver and kidneys due to reticuloendothelial system uptake. The phosphorothioate-modified poly-G tail provides partial nuclease resistance, extending tissue residence time compared to unmodified ODNs. In contrast, the central phosphodiester palindrome allows rapid endosomal acidification and TLR9 triggering. After uptake by pDCs, ODN 2216 accumulates in early endosomes and then redistributes to lysosomal compartments where TLR9 is localized. Urinary excretion is the major elimination pathway for ODN metabolites.
|
| Toxicity/Toxicokinetics |
In preclinical toxicity assessments, ODN 2216 is generally well-tolerated at immunomodulatory doses (10-50 mg/kg in mice). High doses may cause cytokine-mediated toxicities including fever, hypotension, and transient lymphopenia, consistent with TLR9 agonist class effects. In repeat-dose studies, mild hepatocyte vacuolation and transient elevations in liver transaminases have been observed, attributed to immune stimulation rather than direct hepatotoxicity. No genotoxicity or carcinogenicity has been reported for class A CpG ODNs at therapeutic doses. The FITC label does not introduce additional toxicity beyond that of the parent ODN. Careful dose titration is recommended for in vivo experiments to balance immune activation against tolerability.
|
| References | |
| Additional Infomation |
FITC-labeled ODN 2216 is a research tool for studying CpG ODN cellular uptake, intracellular trafficking to TLR9-containing endolysosomal compartments, and TLR9-mediated signaling. The unlabeled ODN 2216 is widely used in immunology research as a human-specific TLR9 agonist for studying pDC activation, type I interferon biology, and vaccine adjuvant development. It is also used in breast cancer and systemic lupus erythematosus research. ODN 2216 has not received regulatory approval for human therapeutic use; it is an investigational research compound. The FITC label enables real-time visualization and quantification of ODN distribution and cellular uptake using fluorescence-based techniques such as confocal microscopy and flow cytometry.
|
| Related CAS # |
ODN 2216;332437-00-0;Biotin-labeled ODN 2216 sodium
|
|---|---|
| Appearance |
White to off-white solid powder
|
| HS Tariff Code |
2934.99.9001
|
| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
|
| Solubility (In Vitro) |
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
|
|---|---|
| Solubility (In Vivo) |
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.
Injection Formulations
Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)(e.g. IP/IV/IM/SC) *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). View More
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)] Oral Formulations
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). View More
Oral Formulation 3: Dissolved in PEG400 (Please use freshly prepared in vivo formulations for optimal results.) |
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
Products are for research use only; We do not sell to patients
Copyright 2020 InvivoChem LLC | All Rights Reserved
