CEMT

Guidelines (The following are our recommended protocols; this is a guideline and should be modified according to your specific needs).
1. Stock Solution Preparation
1.1 Solvent: For most dyes, organic solvents such as anhydrous DMSO are typically used for dissolution.
1.2 Concentration Recommendation: Generally, a high concentration of 1-10 mM stock solution is recommended.
1.3 Storage Conditions: Store at -20 ℃ or -80 ℃ protected from light and avoid repeated freeze-thaw cycles.
2. Working Solution Preparation
2.1 Diluent: Serum-free culture medium or PBS is typically used. Proteins and esterases in serum may interfere with staining or cause dye hydrolysis.
2.2 Working Concentration: 10 μM
Note: Please adjust the dye working solution concentration according to your actual situation and prepare fresh before use.
3. Cell Staining
3.1 Culture cells on sterile coverslips.
3.2 Remove the coverslips from the culture medium and aspirate excess medium.
3.3 Add working solution, gently shake to completely cover the cells, and incubate at 37 ℃ for 20 min.
3.4 Aspirate the dye working solution, wash 2-3 times with culture medium, 5 minutes each time, and observe using a fluorescence microscope or flow cytometry.
Note: If flow cytometry is required, the cells should be resuspended with trypsin before staining.

CEMT (10 μM) exhibits a linear ratiometric fluorescence response to carboxylesterase concentrations from 0 to 15 μg/mL in cell-free 40 mM B-R buffer (pH 7.42) at 37 °C, and its CE-mediated hydrolysis product HMT exhibits a linear ratiometric fluorescence response to pH values from 4.98 to 9.07^[1].
CEMT (10 μM; 20 min) intracellular hydrolysis product HMT exhibits strong mitochondrial colocalization in HepG2 cells, with minimal leakage even under acidic (pH 4.98) conditions, confirming CEMT's ability to lock to mitochondria for in situ imaging^[1].