GW4869 (GW69A) is a selective and non-competitive inhibitor of neutral sphingomyelinase N-SMase (IC50=1 μM). GW4869 also inhibits exosome synthesis/release and is commonly used in exosome-related studies.Usually prepared as a suspension for experiments.

SMase:1 μM

方法：人乳腺癌细胞 MCF-7 用 GW4869 (10 μM) 预处理 30 min，再加入 TNF (3 nM) 孵育 6-24 h，使用 E. coli diacylglycerol kinase assay 检测神经酰胺水平。
结果：GW4869 显著抑制了 TNF 诱导的神经酰胺积累。GW4869 对 N-SMase 有抑制作用。[1]
方法：巨噬细胞 RAW264.7 用 GW4869 (10-20 μM) 预处理 2 h，随后用 LPS (1μg/mL) 孵育 24 h，检测 AchE 活性。
结果：用 10μM GW4869 预处理巨噬细胞后，LPS 触发的外泌体生成在巨噬细胞中显著减弱，AChE的 活性降低了 22%。用 20μM GW4869 处理进一步增强了这种衰减。[2]

方法：为检测体内活性，将 GW4869 (2.5 μg/g) 腹腔注射给 C57BL/6 小鼠，1 h 后注射 LPS (25 μg/g)。
结果：GW4869 预处理小鼠可以减弱 LPS 触发的血液中外泌体和促炎细胞因子的产生，从而减少心肌炎症。[2]
方法：为检测体内活性，将 GW4869 (200 μL 0.3 mg/mL，2-2.5 μg/g) 腹腔注射给 5XFAD 小鼠，每两天一次，持续六周。
结果：GW4869 通过阻止外泌体分泌来减少体内淀粉样斑块的形成。[3]

B. cereu sphosphatidylcholine-PLC is incubated in the presence or absence of 10 μM GW4869 in a reaction mixture containing 100 mM Tris, pH 7.2, 25% glycerol, 20 mM p-nitrophenyl/phosphorylcholine, and production of p-nitrophenol is quantified spectrophotometrically at 410 nm. Protein phosphatase 2A from bovine kidney is incubated in the presence or absence of GW4869 in buffer containing 50 mM Tris, pH 7.4, 1 mM dithiothreitol, 100 μM MnCl2, and 20% glycerol, and phosphatase activity is measured[1].

GW4869 is routinely stored at ?80?°C as a 1.5 mM stock suspension in Me2SO. Right before use, the suspension is solubilized by the addition of 5% methane sulfonic acid (MSA) (2.5 μl of 5% MSA in sterile double-distilled Water are added to 50 μL of GW4869 stock suspension). Cells are treated with GW4869 for 30 min and then TNF is added in 10 μL/well. At the indicated time points, 25 μL of MTT stock solution are added to each well and incubated at 37?°C in 5% CO2 for 3 h. The cell viability is using the MTT assay[1].