Mito-CS1

Guide (The following is our recommended protocol. This protocol is for guidance only and should be modified according to your specific needs).

1. Stock Solution Preparation
1.1 Solvent: DMSO.
1.2 Concentration recommendation: 1 mM.

2. Working Solution Preparation
2.1 Diluent: DPBS, DMEM without phenol red, cell culture medium, or HBSS without calcium chloride, magnesium chloride, magnesium sulfate, sodium bicarbonate, or phenol red (pH 7.1).
2.2 Working concentration: 500 nM, 5 μM, or 10 μM.
2.3 Note: Adjust working solution concentration as needed; prepare fresh before use.

3. Staining Procedure
3.1 Sample type descriptions
3.1.1 For adherent cells^[1][2][3]: HEK 293T cells, human fibroblasts, and copper-deficient fibroblasts with mutant SCO1 and SCO2; plated on poly-L-lysine-coated glass coverslips or chambered coverglass slides 2 days prior to imaging (no trypsinization required for staining).
3.2 Incubation conditions
3.2.1 For adherent cells: Incubate with Mito-CS1 working solution at 37°C, 5% CO₂ for 15-35 min; protect samples from light during incubation. For 10 μM working solution, mix 1:1 v/v with F-127 Pluronic acid prior to incubation.
3.3 Washing steps
3.3.1 For adherent cells: Transfer coverslips to fresh DPBS, or exchange media for fresh HBSS, to remove excess dye.

4. Controls
4.1 Positive control: Incubate cells with CuCl₂ in growth medium for 18 h prior to staining to elevate mitochondrial Cu⁺ levels.
4.2 Negative control: Incubate cells with 100 μM bathocuproine disulfonate (BCS) in growth medium for 12 h or 18 h prior to staining to reduce mitochondrial Cu⁺ levels; use copper-deficient fibroblasts with mutant SCO1 and SCO2 to assess mitochondrial copper pool perturbations.
4.3 Blank control: Exclude Mito-CS1 to rule out reagent fluorescence interference.
4.4 Mitochondrial membrane potential control: Stain cells with 100 nM Rhodamine 123 (HY-D0816) under identical experimental conditions to confirm fluorescence changes are copper-responsive, not due to altered membrane potential.
4.5 Colocalization control: Co-stain cells with 50 nM MitoTracker Deep Red (mitochondrial marker), 2.25 μM BODIPY FL C₅-ceramide-BSA complex (Golgi marker), or 50 nM/250 nM LysoTracker Green DND-26 (lysosomal marker) to confirm mitochondrial localization.
4.6 Viability control: Co-stain cells with 5 μM Hoechst 33342 (HY-15559) to verify cell viability.

5. Detection & Analysis
5.1 Instrument type: Confocal laser-scanning microscope, confocal fluorescence microscope.
5.2 Ex/Em wavelengths:
5.2.1 Excitation at 543 nm, emission range 554-650 nm;
5.2.2 Excitation at 550 nm, emission at 558 nm;
5.3 Result analysis
5.3.1 Fluorescence intensity: Green fluorescence intensity increases with elevated mitochondrial Cu⁺ levels and decreases with reduced mitochondrial Cu⁺ levels; produces a reversible turn-on response upon binding to endogenous exchangeable mitochondrial Cu⁺.