NR-HOCl-TFMU

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

1. Stock Solution Preparation
1.1 Solvent: DMF.
1.2 Recommended Concentration: It is generally recommended to prepare a high-concentration stock solution of 1-10 mM.

2. Working Solution Preparation
2.3 Precautions: Adjust the working solution concentration according to requirements; prepare freshly before use.

3. Staining Procedures
3.1 Sample Type Description
3.1.1 Adherent cells (RAW 264.7 macrophages)^[1][2]:
3.1.1.1 Trypsin digestion is not required.
3.1.2 Suspension cells (HL-60 acute myeloid leukemia cells, K-562 chronic myeloid leukemia cells)^[1][2]:
3.1.2.1 Centrifugation is not required.
3.1.3 Tissue samples (ex vivo tumor sections from local acute myeloid leukemia animal models)^[1]:
3.2 Incubation Conditions
3.2.1 Adherent cells (RAW 264.7 macrophages):
3.2.1.1 Incubate with 5-10 μM NR-HOCl-TFMU for 30 min; for the stimulation group, pretreat with 1 μg/mL LPS (HY-D1056A1) + PMA (HY-18739) for 4 h before dye incubation.
3.2.2 Suspension cells (HL-60/K-562 cells):
3.2.2.1 Incubate with 5-10 μM NR-HOCl-TFMU for 30 min; for the inhibitor group, pretreat with 200 μM 4-ABAH or 1 mM NAC (HY-B0215) for 2 h before dye incubation.
3.2.3 In vivo local tumor model:
3.2.3.1 Intratumorally inject 100 μM NR-HOCl-TFMU.
3.3 Washing Step: Washing is not required for cell and tissue imaging.

4. Control Setup
4.1 Negative Controls:
4.1.1 Untreated cells (blank group).
4.1.2 K-562 cells that do not produce HOCl, incubated with NR-HOCl-TFMU.
4.1.3 HL-60 cells pretreated with MPO inhibitor (4-ABAH) or ROS scavenger (NAC) before dye incubation.
4.1.4 K-562-Luc2 tumors that do not produce HOCl, injected with NR-HOCl-TFMU.
4.1.5 Untreated RAW 264.7 cells without NR-HOCl-TFMU addition.
4.2 Positive Controls:
4.2.1 RAW 264.7 cells stimulated with LPS and PMA, then incubated with NR-HOCl-TFMU.
4.2.2 HL-60 cells incubated with NR-HOCl-TFMU.

5. Detection and Analysis
5.1 Instrument Types:
5.1.1 Confocal fluorescence microscope.
5.1.2 In vivo fluorescence imaging system.
5.2 Excitation/Emission Wavelengths:
5.2.1 For NR₆₆₆-MSA:
5.2.1.1 Confocal fluorescence microscope: Ex: 666 nm, Em: 698 nm.
5.2.1.2 In vivo fluorescence imaging system: Ex: 675 nm, Em: 760 nm.
5.2.2 For TFMU:
5.2.2.1 Confocal fluorescence microscope: Ex: 387 nm/405 nm, Em: 510 nm.
5.3 Result Analysis:
5.3.1 Changes in Fluorescence Intensity:
5.3.1.1 Significant fluorescence enhancement of NR₆₆₆-MSA (near-infrared) and TFMU is observed only in HOCl-producing cells/tissues; no fluorescence enhancement is detected in samples that do not produce HOCl or are pretreated with inhibitors.
5.3.1.2 The near-infrared fluorescence of stimulated RAW 264.7 cells increases by 9.8-fold, and the TFMU fluorescence increases by 1.9-fold.
5.3.1.3 The near-infrared fluorescence of HL-60 cells increases by 5.5-fold, and the TFMU fluorescence increases by 1.5-fold.
5.3.2 Fluorescence Localization:
5.3.2.1 Both NR₆₆₆-MSA and TFMU fluorescence localize inside cells.
5.3.2.2 Fluorescence in ex vivo HL-60-Luc2 tumor sections localizes within the tumor.
5.3.3 Fluorescence Color:
5.3.3.1 NR₆₆₆-MSA produces near-infrared red fluorescence.
5.3.3.2 TFMU produces yellow/green or blue fluorescence, depending on the detection conditions.
5.3.4 Correlation: The fluorescence intensity of NR₆₆₆-MSA is positively correlated with the cargo release amount of TFMU.

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