JC-1

JC-1 (CBIC2) is a fluorescent lipophilic cyanine dye. JC-1 is used to measure mitochondrial membrane potential. When the mitochondrial membrane potential is high, JC-1 aggregates into polymers within the matrix, emitting red fluorescence (λex=585 nm, λem=590 nm). When the mitochondrial membrane potential is low, JC-1 fails to aggregate within the mitochondrial matrix and exists as monomers, emitting green fluorescence (λex=510 nm, λem=527 nm).

α-synuclein:2.6 μM (Kd)

Methods: Primary cells from 12 AML patients (5 newly diagnosed, 7 relapsed) + 5 cell lines were treated with JC-1 (0.1 μM), Annexin V-FITC/PI assay kit, and cytarabine (10⁻⁵ M) + homoharringtonine (HHT, 1500 ng/mL) for apoptosis induction. After 48 hours of co-treatment, P-gp activity was compared between the two Methods via flow cytometry.
Results: Following apoptosis induction, JC-1 red fluorescence remained significantly correlated with Rh 123 activity (P=0.01), whereas green fluorescence lost correlation (P=0.5), demonstrating red fluorescence as the superior marker for assessing P-gp activity.[1]

Instructions
I. Solution preparation
1. Stock solution preparation: JC-1 is dissolved in DMSO to prepare a stock solution. The concentration of the stock solution is usually 5 mg/ml. Please refer to the manufacturer's instructions for the specific concentration.
2. Working concentration: The working concentration of JC-1 is usually 1-20 µg/ml, but the optimal concentration may need to be optimized depending on the cell type and experimental conditions.
3. Dilution: Dilute the stock solution with cell culture medium or appropriate buffer before use.
II. Staining cells
1. Cell preparation: Culture the cells to be tested under appropriate conditions (e.g., 37°C, 5% CO₂).
2. Incubation with JC-1: Add the diluted JC-1 working solution to the cells. Depending on the cell type and experimental conditions, incubate for 15-30 minutes.
3. Washing: After incubation, wash the cells 1-2 times with preheated cell culture medium or PBS to remove excess dye.
3. Fluorescence detection
1. Fluorescence microscopy: Use a fluorescence microscope equipped with appropriate filters to detect green (monomer JC-1) and red (aggregate JC-1) fluorescence. The excitation wavelength of green fluorescence is usually 485-490 nm, and that of red fluorescence is 525-530 nm, with emission peaks at 535 nm (green) and 590 nm (red), respectively.
2. Flow cytometry: Flow cytometry can also be used for quantitative analysis. Use appropriate filters (e.g., 525/40 nm for green fluorescence and 585/42 nm for red fluorescence) for detection.
3. Data interpretation: An increase in red fluorescence indicates a higher mitochondrial membrane potential, while an increase in green fluorescence indicates a loss or decrease in membrane potential.

The above information is based on published literature. Experimental procedures should be appropriately modified to meet specific research demands.

DMSO: 14 mg/mL (21.46 mM), Sonication is recommended.