Biotin-V

Operating Instructions
(The following is a recommended experimental protocol for guidance only; adjust according to your specific requirements)

1. Stock Solution Preparation
1.1 Solvent: Methanol.
1.2 Recommended concentration: 10 μM.

2. Working Solution Preparation
2.1 Serum-free medium or PBS is generally used. Proteins and esterases in serum may interfere with staining results or cause dye hydrolysis.
2.2 Working concentrations: 2.0 μM; 5 μM; 10 μM.
2.3 Notes: Adjust the working solution concentration as needed; prepare fresh before use.

3. Staining Procedures
3.1 Sample types: Cell samples and tumor tissue samples.
3.1.1 Adherent cells (HeLa, 4T1, HepG2, RAW264.7, A549, 3T3): No trypsin digestion is required before staining.
3.1.2 Suspension cells: Not mentioned (no relevant literature sources, thus omitted)
3.1.3 Tumor tissues: Tumor tissues from mouse tumor-bearing models.
3.2 Incubation conditions:
3.2.1 Adherent cancer cells/normal cells: Incubate with 2.0 μM Biotin-V at 37°C for 30 min, protected from light as required for fluorescence imaging.
3.2.2 Viscosity-stimulated HeLa cells: Pre-treat with 10 μM nystatin or monensin for 60 min first, then incubate with 5 μM Biotin-V at 37°C for 30 min, protected from light as required for fluorescence imaging.
3.2.3 In vivo mouse model: Intraperitoneally inject 100 μL of 5 μM Biotin-V; incubation time is determined by the imaging timeline.

4. Control Setup
4.1 Negative control: Biotin receptor-negative normal cells (RAW264.7 macrophages, 3T3 cells) stained under the same conditions.
4.2 Blocking control: Pre-treat HeLa cells with 1 mM D-biotin for 60 min before incubation with 2.0 μM Biotin-V.
4.3 Blank control: Normal mice injected with Biotin-V, used to exclude non-targeted fluorescence.

5. Detection and Analysis
5.1 Instruments: Laser confocal fluorescence microscope; fluorescence lifetime imaging microscope.
5.1.1 Excitation/emission wavelengths for cell imaging: λ_ex=561 nm, λ_em=570-620 nm (corresponding to the red channel for Biotin-V); λ_ex=488 nm, λ_em=500-550 nm (corresponding to the green channel for Mito Tracer Green).
5.1.2 Excitation/emission wavelengths for fluorescence lifetime imaging (FLIM): Pulsed excitation at 561 nm, fluorescence detected at 575-605 nm.
5.1.3 Excitation wavelength for solution spectrum measurement: λ_ex=540 nm.
5.2 Result analysis:
5.2.1 Changes in fluorescence intensity: In solution systems, fluorescence intensity increases by approximately 80-fold when viscosity rises; fluorescence intensity increases by approximately 2-fold in HeLa cells pre-treated with nystatin and by approximately 2.5-fold in HeLa cells pre-treated with monensin; fluorescence intensity in tumor-bearing mice is 4-fold higher than that in normal mice.
5.2.2 Fluorescence localization: Localizes to mitochondria in cancer cells, verified by co-localization with Mito Tracer Green (Pearson's co-localization coefficient=0.89); selectively accumulates in biotin receptor-positive cancer cells and tumor tissues.
5.2.3 Color change: Emits distinct red fluorescence under high viscosity conditions, while fluorescence is negligible in low viscosity environments.
5.2.4 Changes in fluorescence lifetime: When solution viscosity rises, the lifetime extends from 0.514 ns to 3.02 ns; in HeLa cells, the average lifetime extends from 0.813 ns to 1.33 ns after nystatin treatment and to 1.57 ns after monensin treatment; the fluorescence lifetime of tumor tissues (1.468 ns) is longer than that of liver tissues (1.167 ns).

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