Neutrophil isolation protocol.
Neutrophil isolation protocol.
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DOI：
10.3791/745
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发表时间：
2008-07-23
期刊：
影响因子：
--
通讯作者：
Diamond, Scott
中科院分区：
文献类型：
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作者：
Oh, Hana;Siano, Brian;Diamond, Scott
中性粒细胞(PMN)是人类体内含量最丰富的白细胞，也是最早到达炎症免疫反应部位的细胞之一。由于中性粒细胞在炎症中的关键作用，中性粒细胞的运动、细胞因子的产生、吞噬和肿瘤细胞的战斗等功能被广泛研究。为了确定中性粒细胞的特定功能，在体外研究中需要一种干净、快速和可靠的方法将其与其他血细胞分离，特别是因为中性粒细胞寿命很短，应该在采集后2-4小时内使用。在这里，我们展示了一种标准的密度梯度分离方法，使用商业上可用的分离介质从全血中分离人中性粒细胞，分离介质是偏甲酸钠和葡聚糖500的混合物。该程序包括将全血分层在密度梯度介质上，离心，分离中性粒细胞层，以及裂解残留的红细胞。然后，细胞被清洗、计数，并在缓冲液中重新悬浮到所需的浓度。如果操作正确，这种方法已经被证明可以产生>；95%的中性粒细胞样本和>；95%的活力。
Neutrophil polymorphonuclear granulocytes (PMN) are the most abundant leukocytes in humans and among the first cells to arrive on the site of inflammatory immune response. Due to their key role in inflammation, neutrophil functions such as locomotion, cytokine production, phagocytosis, and tumor cell combat are extensively studied. To characterize the specific functions of neutrophils, a clean, fast, and reliable method of separating them from other blood cells is desirable for in vitro studies, especially since neutrophils are short-lived and should be used within 2-4 hours of collection. Here, we demonstrate a standard density gradient separation method to isolate human neutrophils from whole blood using commercially available separation media that is a mixture of sodium metrizoate and Dextran 500. The procedure consists of layering whole blood over the density gradient medium, centrifugation, separation of neutrophil layer, and lysis of residual erythrocytes. Cells are then washed, counted, and resuspended in buffer to desired concentration. When performed correctly, this method has been shown to yield samples of >95% neutrophils with >95% viability.