Lys-VBOD

Guide (The following is our recommended protocol. This protocol is for guidance only and should be modified according to your specific needs).

1. Stock Solution Preparation
1.1 Solvent: Glycerol/water mixtures for in vitro assays; 1640 medium for cell assays.
1.2 Concentration recommendation: 10 μM.

3. Staining Procedure
3.1 Sample type: Adherent cells (Bel-7402 and A549 cell lines).[1]
3.1.1 Adherent cells: Split cultured cells into 35 mm diameter confocal dishes at a density of ~3×10⁵ cells per dish, then incubate in 1640 medium for 24 h before staining.
3.2 Incubation conditions: Incubate cells with 10 μM Lys-VBOD at 37°C for 30 min.
3.3 Washing steps: Wash cells three times with PBS to remove free probe.

4. Controls
4.1 Set up the following controls:
4.1.1 VBOD control: Perform viscosity titration under identical conditions to Lys-VBOD to assess the effect of the morpholine group on fluorescence properties.
4.1.2 BOD−OH control: Incubate cells with BOD−OH under identical conditions to Lys-VBOD to confirm that fluorescence intensity increases are due to viscosity changes rather than probe accumulation.
4.1.3 Co-localization control: Co-stain cells with commercial Lyso-Tracker Green to validate lysosomal targeting of Lys-VBOD.

5. Detection & Analysis
5.1 Instrument: Confocal laser scanning microscopy for cell imaging; spectrofluorometer for in vitro fluorescence measurements.
5.1.1 Excitation wavelength (λabs): 602 nm; emission wavelength (λem): 637 nm.
5.2 Result analysis:
5.2.1 Fluorescence intensity changes: Fluorescence intensity increases significantly with rising viscosity, showing a linear relationship between log(fluorescence intensity) and log(viscosity); fluorescence intensity increases gradually over time in Dexamethasone (HY-14648)-stimulated cells.
5.2.2 Fluorescence localization: Red fluorescence localizes specifically to lysosomes, confirmed by co-localization with Lyso-Tracker Green (Pearson’s colocalization coefficient = 0.91).
5.2.3 Color changes: Weak fluorescence in low viscosity environments; bright red fluorescence in high viscosity environments.
5.2.4 Fluorescence lifetime: Fluorescence lifetime lengthens with increasing viscosity, showing a positive correlation with viscosity levels.

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