BOD-Tz-TMR

Operating Instructions (The following is a recommended experimental protocol for guidance only, and should be adjusted according to your specific requirements)

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.
2.2 Working Concentration: 5 μM.
2.3 Notes: Adjust the working solution concentration according to requirements; prepare and use the solution immediately.

3. Staining Procedures
3.1 Sample Type Description
3.1.1 Adherent cells (RAW 264.7 cells, HeLa cells)^[1][2]:
3.1.1.1 Trypsin digestion is not required for metabolic labeling; after labeling, paraformaldehyde fixation can be used to confirm stability.
3.2 Incubation Conditions
3.2.1 Pre-labeling: Incubate RAW 264.7 cells with 20 μM Ac₄ManN-BCN for 24 h to perform glycometabolic engineering modification.
3.2.2 Labeling: Incubate the modified cells with 5 μM BOD-Tz-TMR for 2 h to perform bioorthogonal labeling.
3.2.3 NO Stimulation (in vitro): For detection of exogenous NO, add aqueous NO solution to the labeled cells; for detection of endogenous NO, stimulate the labeled RAW 264.7 cells with 20 μg/mL ox-LDL, or 20 μg/mL LPS + 50 μg/mL L-Arg + 150 U/mL IFN-γ, or 2.67 × 10⁶ CFU/mL S. aureus for 24 h.
3.3 Washing Step: Wash the fixed cells subjected to two-step staining three times with PBS.

4. Control Setup
4.1 Set up negative controls: labeled cells without stimulation.
4.2 Set up one-step staining controls: Incubate cells with pre-reacted BOD-Tz-TMR + Ac₄ManN-BCN for 24 h to verify the stability of the two-step labeling.
4.3 Set up positive controls to validate the effectiveness of the experimental system.
4.4 Set up blank controls to exclude fluorescent interference from reagents.

5. Detection and Analysis
5.1 Instrument Types: Confocal fluorescence microscope, fluorescence microscope, or in vivo/ex vivo imaging system.
5.2 Excitation/Emission Wavelengths
5.2.1 Green channel (BOD component): λₑₓ = 488 nm, λₑₘ = 500-550 nm.
5.2.2 Red channel (TMR component): λₑₘ = 570-620 nm.
5.2.3 Nile Red (plaque staining): λₑₓ = 530 nm, λₑₘ = 620-660 nm.
5.3 Result Analysis
5.3.1 Changes in fluorescence intensity:
5.3.1.1 After bioorthogonal labeling: The fluorescence intensity of the green channel increases significantly, while that of the red channel remains at a low level.
5.3.1.2 After exposure to NO: The fluorescence intensity of the red channel increases significantly, while that of the green channel decreases.
5.3.1.3 In the aorta of atherosclerotic mice, the red fluorescence intensity increases with plaque progression; both the red channel fluorescence intensity and the red/green fluorescence intensity ratio of advanced atherosclerotic plaques are higher than those of early plaques.
5.3.2 Fluorescence localization: BOD-Tz-TMR distributes throughout the cell after labeling; it localizes in the cytoplasm of labeled cells.
5.3.3 Color change: In the absence of NO, green fluorescence from the BOD component is observed; in response to NO, red fluorescence from the TMR component is observed.
5.3.4 Ratio analysis: Calculate the ratio of red channel fluorescence intensity to green channel fluorescence intensity (I_Red/I_Green); within the range of 0-60 μM, this ratio increases linearly with the increase of NO concentration, and the ratio of NO-stimulated cells or atherosclerotic plaque samples is significantly higher than that of the control group.
5.3.5 In vivo/ex vivo imaging: The in vivo probe accumulates at atherosclerotic plaque sites in ApoE^-/^- mice, and colocalizes with Nile Red (HY-D0718) staining.

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