HOTPy

Guidelines (The following is a recommended experimental protocol for guidance only; adjustments are required based on your specific needs)

1. Stock Solution Preparation
1.2 Recommended concentration: 40 μM.

2. Working Solution Preparation
2.1 Diluent: Phosphate-buffered saline (PBS, 10 mM, pH 7.4).
2.2 Working concentration: 10 μM.
2.3 Notes: Adjust the working solution concentration as needed; prepare and use immediately.

3. Staining Procedure
3.1 Sample Type Description
3.1.1 Adherent cells[1]: Human liver cells (LO2 cells); trypsin digestion is not required before staining.
3.2 Incubation Conditions
3.2.1 Adherent cells: Incubate with 10 μM HOTPy at 37°C for 30 min.
3.3 Washing Steps
3.3.1 Adherent cells: After incubation, wash 3 times with 1 mL PBS at room temperature.

4. Control Setup
4.1 Cell Imaging Controls:
4.1.1 Negative control: LO2 cells without HOTPy incubation.
4.1.2 Positive control: LO2 cells pretreated with Monensin (HY-N4302) (10 μM), Nystatin (HY-17409) (10 μM) or ethanol (0.8 M) for 60 min before HOTPy incubation.

5. Detection and Analysis
5.1 Instrument type: Confocal laser scanning microscope.
5.2 Excitation/Emission Wavelengths
5.2.1 Cell imaging: Ex = 430 nm, Em = 600-700 nm (corresponding to the red channel).
5.3 Result Analysis
5.3.1 Fluorescence intensity change: An increase in red fluorescence intensity indicates elevated mitochondrial viscosity in cells or liver tissues; a decrease in fluorescence intensity indicates reduced viscosity.
5.3.2 Fluorescence localization: Red fluorescence shows a punctate distribution in mitochondria of LO2 cells.