C-TPA

Operating Instructions (The following is a recommended experimental protocol for guidance only; please adjust it according to your actual needs)
1. Stock Solution Preparation
1.1 Solvent: For most dyes, organic solvents are commonly used for dissolution, such as anhydrous DMSO.
1.2 Concentration Recommendation: It is generally recommended to prepare a high-concentration stock solution of 1-10 mM.
2. Working Solution Preparation
2.1 Diluent: Serum-free medium or PBS is usually used. Proteins and esterases in serum may interfere with staining results or cause dye hydrolysis.
2.2 Working Concentration: 10 μM for cell imaging; 15 μM for tissue imaging.
2.3 Notes: The concentration of the working solution can be adjusted as needed; it must be prepared and used immediately.
3. Staining Procedure
3.1 Sample Types^[1]:
3.1.1 Adherent cells: 4T1 mouse breast cancer cells, 3T3 mouse embryonic normal cells, A549 human lung cancer cells, HL7702 human normal liver cells.
3.1.3 Tissue samples: Mouse tumor tissues and normal liver tissues.
3.2 Incubation Conditions:
3.2.1 Adherent cells: Incubate with 10 μM C-TPA for 0.5 h.
3.2.2 Tissue samples: Incubate with 15 μM C-TPA.
4. Control Setup
4.1 Cellular level: Use normal cells (3T3 mouse embryonic normal cells, HL7702 human normal liver cells) as controls, and conduct C-TPA staining and imaging comparison with cancer cells (4T1 mouse breast cancer cells, A549 human lung cancer cells) under the same conditions.
4.2 Tissue level: Use mouse normal liver tissues as controls, and conduct C-TPA staining and imaging comparison with mouse tumor tissues under the same conditions.
4.3 Colocalization control: Use the lipid droplet dye Nile Red (HY-D0718) as a positive control for lipid droplet localization, and perform co-staining and colocalization analysis with C-TPA.
5. Detection and Analysis
5.1 Instrument: Confocal fluorescence microscope.
5.1.1 Cell imaging: Ex = 405 nm, Em = 425-475 nm; Tissue imaging: Ex = 405 nm, Em = 500-550 nm.
5.2 Result Analysis:
5.2.1 Fluorescence intensity change: The green fluorescence intensity of C-TPA in cancer cells (4T1, A549) is significantly higher than that in normal cells (3T3, HL7702); the green fluorescence intensity of C-TPA in tumor tissues is stronger than that in normal liver tissues.
5.2.2 Fluorescence localization: C-TPA specifically accumulates in lipid droplets (LDs) of cells, which is confirmed by colocalization experiments with Nile Red (Mander's overlap coefficient = 0.93, Pearson's colocalization coefficient = 0.89).
5.2.3 Color change: It emits bright green fluorescence in low-polarity environments (lipid droplets of cancer cells, tumor tissues) and weak green fluorescence in high-polarity environments (lipid droplets of normal cells, normal tissues).