TPE-Ph-In

Operating Guidelines (The following is a recommended experimental protocol for guidance only, and adjustments are required based on your specific needs)
1. Stock Solution Preparation
1.1 Solvents: DMSO, aqueous solution or cell culture medium are optional.
1.2 Concentration recommendation: 100 mM; note that there is no specified stock solution concentration when preparing with aqueous solution as the solvent.
2. Working Solution Preparation
2.1 Diluents: Cell culture medium, medium or aqueous solution are optional.
2.2 Working concentrations: 4 μM, 5 μM, 10 μM.
2.3 Notes: The concentration of the working solution can be adjusted according to requirements; prepare and use it immediately.
3. Staining Procedures
3.1 Sample Type Description
3.1.1 Mammalian adherent cells^[1][2][4]: Including HeLa, HT22 and ARPE-19 cells; trypsinization is not required before staining for microscopic observation, and only needed when collecting cells for flow cytometry.
3.1.2 Mammalian suspension cells^[1]: Mouse sperm cells.
3.1.3 Plant cells^[3]: Including Arabidopsis thaliana root/leaf cells, tobacco seedling root cells, tobacco BY-2 cells and tobacco BY-2 protoplasts; probe penetration is inconsistent in Arabidopsis thaliana root cells away from the root tip, and the fluorescence signal of tobacco BY-2 protoplasts is enhanced after cell wall digestion.
3.2 Incubation Conditions
3.2.1 Mammalian adherent cells: Incubate with 4-10 μM TPE-Ph-In for 20-30 min in a 37°C, 5% CO₂ incubator.
3.2.2 Mammalian suspension cells: Incubate with 5 μM TPE-Ph-In for 1 h.
3.2.3 Plant cells: Incubate with 10 μM TPE-Ph-In.
3.3 Washing Procedures
3.3.1 Mammalian adherent cells: Wash with PBS 2-3 times, or remove the TPE-Ph-In-containing medium after incubation.
3.3.2 Mammalian suspension cells: No washing step is required during real-time monitoring of ΔΨₘ.
3.3.3 Plant cells: Omit the washing step completely.
4. Control Setup
4.1 Unstained blank cell control: Used to eliminate reagent fluorescence interference.
4.2 Positive control with Oligomycin (HY-N6782)-treated cells: Used to verify the response of mitochondrial ΔΨ_m.
4.3 Negative control with Carbonyl cyanide 3-chlorophenylhydrazone (CCCP) (HY-100941)-treated cells: Used to verify the response of mitochondrial ΔΨ_m.
5. Detection and Analysis
5.1 Instrument types: Confocal microscope, flow cytometer, fluorescence microscope, inverted fluorescence microscope, structured illumination microscope (SIM).
5.2 Excitation/emission wavelengths: Excitation wavelengths include 450 nm, 488 nm, 489 nm, 530 nm; emission wavelengths include 551-656 nm, 660 nm, ~694 nm (red light region).
5.3 Result Analysis
5.3.1 Changes in fluorescence intensity: An increase in red fluorescence intensity corresponds to an increase in mitochondrial ΔΨ_m (treatment with Oligomycin); a decrease in red fluorescence intensity corresponds to a decrease in mitochondrial ΔΨₘ (treatment with CCCP or early apoptosis). Bright red fluorescence in the midpiece of mouse sperm indicates high sperm activity, while weak or no fluorescence indicates low sperm activity.
5.3.2 Fluorescence localization: TPE-Ph-In specifically localizes to mitochondria in mammals and plants; it colocalizes with MitoTracker Green in mammalian cells.
5.3.3 Color change: The dye emits red fluorescence when accumulated in mitochondria.
5.3.4 Morphological and dynamic analysis: Observe mitochondrial morphology (filamentous, rod-shaped, granular) and distribution (uniformly dispersed, aggregated around the nucleus) via fluorescence imaging; quantify mitochondrial length and the distance from mitochondria to the nucleus using image analysis software. SIM imaging can resolve mitochondrial cristae structures and capture the dynamic fission and fusion processes of mitochondria in living plant cells; the fluorescence intensity of tobacco BY-2 protoplasts remains over 68.7% after 400 consecutive frames of shooting.

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