Iwai Kazuya, Yamamoto Satoshi, Yoshida Mitsutaka, Shiba Kiyotaka
Division of Protein Engineering, Cancer Institute, Japanese Foundation for Cancer Research, Tokyo, Japan.
Department of Oral & Maxillofacial Implantology, Tokyo Dental College, Tokyo, Japan.
Methods Mol Biol. 2017;1660:343-350. doi: 10.1007/978-1-4939-7253-1_27.
This chapter describes a method for isolating human salivary extracellular vesicles (EVs) using density gradient ultracentrifugation. Standard protocols established for isolation of EVs from blood or a conditioned medium of cultured cells do not work for whole saliva, due to its viscosity. Therefore, procedures including a pretreatment step and utilizing iodixanol as a gradient material enable EVs to be concentrated to a 1.1 g/ml density. This protocol is compatible with both swing and angle rotors. By employing an angle rotor, which enables high g-force, the centrifugation time was reduced to 4 h from the 17 h required when using a swing rotor.
本章介绍了一种使用密度梯度超速离心法分离人唾液细胞外囊泡（EVs）的方法。由于全唾液的粘度，从血液或培养细胞的条件培养基中分离EVs的标准方案不适用于全唾液。因此，包括预处理步骤并使用碘克沙醇作为梯度材料的程序能够将EVs浓缩至密度为1.1 g/ml。该方案适用于摆动式和角度式转子。通过使用能够产生高离心力的角度式转子，离心时间从使用摆动式转子所需的17小时减少到了4小时。
