Ling E, Shirai K, Kanehatsu R, Kiguchi K
Laboratory of Silkworm Physiology, Department of Applied Biology, Faculty of Textile Science and Technology, Shinshu University, Ueda, Nagano 386-8567, Japan.
Tissue Cell. 2005 Apr;37(2):101-7. doi: 10.1016/j.tice.2004.10.007. Epub 2005 Jan 24.
We have developed a modified method to detect phenoloxidase activity on hemocytes by using freshly prepared l-DOPA (1 mg/ml in 35% ethanol) to fix and incubate larval hemocytes. This method is more sensitive than the common method, in which hemocytes were fixed in 4% formaldehyde and then incubated with 2 mg/ml l-DOPA in water separately. Phenoloxidase assayed using this modified method can be inhibited by phenyltiourea (phenoloxidase inhibitor). After incubation with l-DOPA solution in ethanol, most prohemocytes, all plasmatocytes and young granulocytes are stained brown due to oxidation of l-DOPA into pigments, indicating that they have phenoloxidase. Oenocytoids are dimly stained because many of their cell inclusions have been released during the treatment. Large propidium-iodide-negative prohemocytes have strong phenoloxidase activity and are easily misunderstood as propidium-iodide-positive oenocytoids if the fluorescent method is not used for identification. Thus, in addition to oenocytoids and plasmatocytes, some prohemocytes and granulocytes in the silkworm also have phenoloxidase.
我们开发了一种改进方法，通过使用新制备的左旋多巴（在35%乙醇中为1 mg/ml）固定并孵育幼虫血细胞来检测血细胞中的酚氧化酶活性。该方法比常用方法更灵敏，常用方法是将血细胞固定在4%甲醛中，然后分别与2 mg/ml的左旋多巴水溶液孵育。用这种改进方法测定的酚氧化酶可被苯硫脲（酚氧化酶抑制剂）抑制。在用乙醇中的左旋多巴溶液孵育后，大多数原血细胞、所有浆血细胞和年轻粒细胞因左旋多巴氧化成色素而被染成棕色，表明它们具有酚氧化酶。oenocytoids染色较浅，因为在处理过程中它们的许多细胞内含物已被释放。如果不使用荧光方法进行鉴定，大的碘化丙啶阴性原血细胞具有很强的酚氧化酶活性，很容易被误解为碘化丙啶阳性的oenocytoids。因此，除了oenocytoids和浆血细胞外，家蚕中的一些原血细胞和粒细胞也具有酚氧化酶。
