To ensure optimal experimental reproducibility, please strictly follow the steps below to prepare the stock
solution.
Formulation: Accurately weigh 1.0 mg of OmiFluo™ BOSHY-LD Yellow (MW≈670) or an equivalent
amount of OmiFluo™ BOSHY-LD Green.
Dissolution: Add 298.5 μL of anhydrous DMSO.
Mixing: Vortex thoroughly until completely dissolved.
Final Product: A 5 mM stock solution is obtained.
⚠️ Storage Precautions: The stock solution should be stored at -20°C, protected from light. It
is valid for approximately 6 months. The working solution must be prepared fresh for each use; repeated
freeze-thaw cycles are strictly prohibited.
2. Cell Staining Procedure
Day 1: Cell Plating
Seed HeLa or related cell lines into 35 mm confocal dishes.
Seeding Density: 2-5×10⁴ cells/mL, 1.5 mL per dish.
Culture Conditions: Incubate at 37°C with 5% CO₂ for 24 hours until cell confluence reaches 60-70%.
Day 2: Staining and Imaging
Step 1: Working Solution Preparation
Add 2 μL of the 5 mM stock solution to 998 μL of serum-free medium.
Mix gently to obtain a 10 μM intermediate solution (avoid vigorous vortexing to prevent precipitation).
Dilute again 1:10 with pre-warmed medium to finally obtain a 1 μM working solution.
Step 2: Cell Staining
Aspirate the old medium and gently wash once with pre-warmed PBS.
Add 1 mL of the 1 μM staining working solution.
Place in a 37°C incubator and incubate for 20 minutes, protected from light.
Aspirate the staining solution and wash twice with pre-warmed PBS (1 mL each, 1 minute).
Add 1 mL of fresh complete medium (phenol red-free is preferred).
3. Confocal Imaging Settings
A Zeiss LSM 880 or equivalent confocal microscope is recommended.
Parameter
Recommended Settings
Objective
63× Oil Objective (NA=1.4)
Excitation Wavelength
488 nm (Argon laser, recommended power 5-10%) or 405 nm
Emission Collection (OmiFluo™ BOSHY-LD Yellow)
520 - 620 nm (Green/Yellow Channel)
Emission Collection (OmiFluo™ BOSHY-LD Green)
500 - 550 nm
Resolution
1024×1024 pixels, 12-bit depth
Tip: BOSHY probes feature a large Stokes shift. Please ensure correct emission and collection
bandwidth settings to achieve the best signal-to-noise ratio.