cis-Ned19

cis-Ned19 is an irreversible nicotinic acid adenine dinucleotide phosphate (NAADP) antagonist. cis-Ned19 localizes to lysosomes and endolysosomal vesicles, stains two-pore calcium channels and fluorescently labels NAADP receptors (Ex/Em = 365/410 nm)^[1][2][3]. In Vitro:Guidelines (The following is our recommended protocol; this protocol serves as a guideline only and should be modified to suit your specific needs.)
1. Preparation of Stock and Working Solutions
a. Prepare a 10 mM cis-Ned19 stock solution in anhydrous DMSO.
b. Dilute the stock solution using pre-warmed, serum-free cell culture medium or PBS to prepare a 10 μM cis-Ned19 working solution.
Note: Please adjust the concentration of the cis-Ned19 working solution according to your specific experimental requirements, and prepare it immediately before use.
2. Cell Staining (Suspension Cells)
a. Centrifuge to harvest the cells, then wash twice with PBS (5 minutes per wash). Adjust the cell density to 1×10⁶ cells/mL.
b. Add 1 mL of the cis-Ned19 working solution and incubate at room temperature for 10 minutes.
c. Centrifuge at 400 × g for 3–4 minutes, and discard the supernatant.
d. Wash the cells twice with PBS (5 minutes per wash).
e. Resuspend the cells in 1 mL of serum-free medium or PBS, then observe using a fluorescence microscope or flow cytometer.
3. Cell Staining (Adherent Cells)
a. Culture adherent cells on sterile coverslips.
b. Remove the coverslips from the culture medium and aspirate any excess medium.
c. Add the working solution, gently agitating to ensure it completely covers the cells, and incubate at room temperature for 10 minutes.
d. Aspirate the dye working solution, then wash 2–3 times with culture medium (5 minutes per wash). Observe using a fluorescence microscope or flow cytometer.
Note: If analysis via flow cytometry is required, the cells must be detached using trypsin digestion and resuspended prior to staining.
cis-NED19 (10 μM) binds to sites localized in endolysosomes of human umbilical vein endothelial cells^[1].
cis-Ned19 (10 min) colocalizes significantly with the lysosomal marker LysoTracker but not with endoplasmic reticulum or mitochondrial markers in cultured human umbilical vein endothelial cells^[2].
cis-Ned19 inhibits Ca²⁺ release (IC₅₀ = 800 nM) and [³²P]NAADP binding (IC₅₀ = 15 μM)^[3].