CSP

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: Cell culture medium.
2.2 Working concentration: 1 μM, 5 μM.
2.3 Note: Adjust working solution concentration as needed; prepare fresh before use.

3. Staining Procedure
3.1 Sample type: Adherent cells (HeLa cells), suspension cells (RAW cells).[1]
3.1.1 Adherent cells: No trypsinization mentioned for live cell imaging.
3.2 Incubation conditions: Incubate cells with 1 μM or 5 μM CSP for 30 min; co-incubate with 0.3 μM LysoBrite NIR (LB-NIR) or 0.3 μM MitoTracker Deep Red (MTDR) for an additional 30 min.
3.3 Washing steps: Wash with pH 7.4 PBS three times before exposing to MMP-modulating conditions.

4. Detection & Analysis
4.1 Instrument: Confocal laser scanning microscope.
4.1.1 CSP: Ex = 488 nm, Em = 550-650 nm; LB-NIR/MTDR: Ex = 633 nm, Em = 680-750 nm.
4.2 Result analysis:
4.2.1 At normal mitochondrial membrane potential (MMP), CSP localizes to mitochondria, showing high colocalization coefficient (CLC) with MTDR and low CLC with LB-NIR.
4.2.2 When MMP decreases, CSP migrates to lysosomes, showing increasing CLC with LB-NIR over time.
4.2.3 When MMP recovers, CSP moves back to mitochondria, showing decreasing CLC with LB-NIR.
4.2.4 CSP emits fluorescence with green pseudocolor in imaging experiments.