Rhobo6

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.