Collaborative Research Group, Tokyo Plastic Dental Society, Tokyo 114-0002, Japan.
Division of Anatomy and Cell Biology of the Hard Tissue, Institute of Medicine and Dentistry, Niigata University, Niigata 951-8514, Japan.
Int J Mol Sci. 2021 Jan 21;22(3):1040. doi: 10.3390/ijms22031040.
Polyphosphate (polyP) is released from activated platelets and activates the intrinsic coagulation pathway. However, polyP may also be involved in various pathophysiological functions related to platelets. To clarify these functions, we established a cytochemical method to reproducibly visualize polyP in platelets. Platelets obtained from healthy non-smoking donors were suspended in phosphate-buffered saline and quickly immobilized on glass slides using a Cytospin. After fixation and membrane permeabilization, platelets were treated with 4',6- diamidino-2-phenylindole (DAPI) and examined using a fluorescence microscope with a blue-violet excitation filter block (BV-2A). Fixed platelets were also subjected to immunocytochemical examination to visualize serotonin distribution. Under the optimized conditions for polyP visualization, immobilized platelets were fixed with 10% neutral-buffered formalin for 4 h or longer and treated with DAPI at a concentration of 10 µg/mL in 0.02% saponin- or 0.1% Tween-20-containing Hanks balanced salt solution as a permeabilization buffer for 30 min at room temperature (22-25 °C). Based on the results obtained by using activated platelets, treatment with alkaline phosphatases, and serotonin release, the DAPI targets were identified as polyP. Therefore, this cytochemical method is useful for determining the amount and distribution of polyP in platelets.
多聚磷酸盐（polyP）从活化的血小板中释放出来，并激活内源性凝血途径。然而，polyP 可能也参与与血小板有关的各种病理生理功能。为了阐明这些功能，我们建立了一种细胞化学方法，可重现性地观察血小板中的 polyP。从健康不吸烟的供体获得的血小板悬浮在磷酸盐缓冲液中，并使用细胞离心机快速固定在载玻片上。固定和膜透化后，用 4'，6-二脒基-2-苯基吲哚（DAPI）处理血小板，并使用带有蓝色-紫色激发滤光片块（BV-2A）的荧光显微镜进行检查。固定的血小板还进行免疫细胞化学检查，以观察 5-羟色胺的分布。在优化的 polyP 可视化条件下，将固定的血小板用 10%中性缓冲福尔马林固定 4 小时或更长时间，并在含有 0.02%皂素或 0.1%吐温-20 的 Hank's 平衡盐溶液中的 DAPI 浓度为 10μg/mL 处理 30 分钟，在室温（22-25°C）下。基于使用活化血小板、碱性磷酸酶处理和 5-羟色胺释放获得的结果，DAPI 靶标被鉴定为 polyP。因此，这种细胞化学方法可用于确定血小板中 polyP 的量和分布。
