BTCV-CO

Guidelines (The following is a recommended experimental protocol for guidance only; please adjust it according to your specific needs)

1. Stock Solution Preparation
1.1 Solvent: For most dyes, organic solvents are usually used for dissolution, such as anhydrous DMSO.
1.2 Concentration Recommendation: It is generally recommended to prepare a high-concentration stock solution of 1-10 mM.

2. Working Solution Preparation
2.1 Diluent: PBS buffer (pH 7.4, containing 5% DMSO) is used in literature.
2.2 Working Concentration: 0-50 μM.
2.3 Notes: Adjust the working solution concentration according to requirements; prepare and use it immediately.

3. Staining Procedure
3.1 Applicable to adherent cells.
3.2 Incubation Conditions: First add BTCV-CO (5 μM) and PdCl₂ (5 μM), incubate at 37°C in the dark for 15 min; then add CORM-2 (CO donor), continue incubation at 37°C in the dark for 30 min before imaging.
3.3 Washing Step: After incubation, wash the cells 2-3 times with pre-warmed PBS to remove free probes and reduce background fluorescence.

4. Control Setup
4.1 Unstained Control: Used to adjust instrument voltage and eliminate autofluorescence of cells.
4.2 Negative Control: Cells are incubated only with BTCV-CO + PdCl₂ without adding CORM-2.

5. Detection and Analysis
5.1 Instrument Type: Confocal microscope, fluorescence microscope.
5.2 Excitation/Emission Wavelengths: Excitation wavelength is 476 nm (465 nm for spectral experiments); collect emission around 500-550 nm in the green channel (corresponding to the 546 nm peak), and collect long-wave emission (~710 nm) in the red channel.
5.3 Result Analysis: After BTCV-CO reacts with CO, the fluorescence in the green channel increases significantly, while that in the red channel changes slightly. The I_green/I_red ratio increases with the rise of CO concentration (concentration-dependent). The intracellular CO level can be semi-quantitatively analyzed by dual-channel ratio imaging. The probe has good cell membrane permeability and low cytotoxicity (below 25 μM).