Rhobo6 

Rhobo6 is a cell-impermeable glycan-binding, fluorescence turn-on imaging agent with a K_d of 53 µM for glycans. Rhobo6 reversibly binds target glycans and enables wash-free live ECM visualization. Rhobo6 can be used for fluorescent labeling of ECM in living samples or decellularized tissues (Ex/Em = 488/561 nm)^[1].

Guidelines (The following is our recommended protocol; this protocol serves as a guideline only and should be modified to suit your specific needs.)
1. Preparation of Stock and Working Solutions
a. Prepare a 10 mM Rhobo6 stock solution in anhydrous DMSO.
b. Dilute the stock solution using pre-warmed, serum-free cell culture medium or PBS to prepare a 5 μM Rhobo6 working solution.
Note: Please adjust the concentration of the Rhobo6 working solution according to your specific experimental requirements, and prepare it immediately before use.
2. Cell Staining (Suspension Cells)
a. Centrifuge to harvest the cells, then wash twice with PBS (5 minutes per wash). Adjust the cell density to 1×10⁶ cells/mL.
b. Add 1 mL of the Rhobo6 working solution and incubate at room temperature for 1 hour.
c. Centrifuge at 400 × g for 3–4 minutes, and discard the supernatant.
d. Wash the cells twice with PBS (5 minutes per wash).
e. Resuspend the cells in 1 mL of serum-free medium or PBS, then observe using a fluorescence microscope or flow cytometer.
3. Cell Staining (Adherent Cells)
a. Culture adherent cells on sterile coverslips.
b. Remove the coverslips from the culture medium and aspirate any excess medium.
c. Add the working solution, gently agitating to ensure it completely covers the cells, and incubate at room temperature for 1 hour.
d. Aspirate the dye working solution, then wash 2–3 times with culture medium (5 minutes per wash). Observe using a fluorescence microscope or flow cytometer.
Note: Rhobo6 should only be used for live cell staining. If analysis via flow cytometry is required, the cells must be detached using trypsin digestion and resuspended prior to staining.
Rhobo6 (5 μM) turns on and red shifts upon binding diols in cell-free solution, with enhanced molar absorptivity, quantum yield, and a fluorescence contrast of 7.3 using 561 nm excitation and 575 nm emission detection^[1].
Rhobo6 (5 μM) exhibits broad binding specificity for glycans in a cell-free array assay, with reduced binding to negatively charged glycans^[1].
Rhobo6 (5 μM; 1 h) labels purified ECM components in vitro in a glycan-dependent manner, with broad coverage of fibrous, network-forming, proteoglycan, and polysaccharide ECM constituents^[1].
Rhobo6 (1-50 μM; 3 h) binds to collagen I gels in vitro with an apparent K_d of 53 μM, reaching near-maximum signal within 60 min at 5 μM^[1].
Rhobo6 (5 μM; 9.6 h) exhibits negligible photobleaching when bound to aggrecan-coated substrates in vitro over extended imaging periods^[1].
Rhobo6 (5 μM; 1 h at 37 °C) labels glycan-rich cell surfaces of MUC1ΔCT-expressing MCF10A cells in a glycan-dependent manner, with distinguishable fluorescence lifetimes for free and bound dye populations^[1].
Rhobo6 (5 μM; 48 h) labels ECM and tracks ECM remodeling during 4T1 breast cancer spheroid invasion in vitro without impairing invasive potential, revealing fiber orientation changes associated with invasion^[1].

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

1. In vivo Imaging
a. Take 10 mM Rhobo6 (dissolved in DMSO) stock solution and dilute to 1 mM working solution with sterile PBS.
b. Sterilize the working solution through a 0.2 μm filter. Use immediately or in single-use aliquots and store at -20 °C, avoiding freeze-thaw cycles and exposure to sunlight.
c. Inject Rhobo6 working solution via the retroorbital vein at a dose of 3.5 mg/kg of animal body weight 15–30 minutes before imaging.
Note: Rhobo6 kinetic studies should be performed for each animal model to determine peak signal time.
Rhobo6 (3.5 mg/kg; i.v. [retro-orbital]; single dose) enables broad, wash-free visualization of ECM structures across most mouse tissues following retro-orbital injection, with no apparent toxicity^[1].
Rhobo6 (i.v. [retro-orbital]; single dose) enables intravital visualization of tumor-associated ECM remodeling, distinguishing altered ECM architecture in breast cancer lesions from normal mammary gland ECM^[1].
Rhobo6 (100 µM; injectable solution; single injection) labels extracellular and luminal structures in C. elegans without intracellular accumulation^[1].
Rhobo6 (5 µM; bath immersion in tank water; single exposure) enables visualization of ECM structures in zebrafish larvae during wound healing when delivered via tail incision^[1].

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

642.23

C₃₅H₂₈B₂N₂O₉

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Room temperature in continental US; may vary elsewhere.

Please store the product under the recommended conditions in the Certificate of Analysis.

• [1]. Fiore A, et al. Live imaging of the extracellular matrix with a glycan-binding fluorophore. Nat Methods. 2025;22(5):1070-1080.