A rapid two-step procedure for the purification of human peripheral blood basophils to near homogeneity
A rapid two-step procedure for the purification of human peripheral blood basophils to near homogeneity
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DOI：
10.1111/j.1365-2222.2007.02919.x
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发表时间：
2008-03-01
影响因子：
6.1
通讯作者：
Falcone, F. H.
中科院分区：
文献类型：
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作者：
Gibbs, B. F.;Papenfuss, K.;Falcone, F. H.
背景嗜碱性粒细胞越来越多地被用作过敏性炎症的指示物和主要的过敏性效应细胞来研究信号通路。因此，我们设计了一种在1.5h内从少量外周血中分离纯化人嗜碱性粒细胞的方法，该方法不需要使用洗脱器等专用设备。肝素化或K3-乙二胺四乙酸血样首先在Hetasep中离心，然后直接使用免疫磁珠进行阴性选择。用胞浆May-Grunwald染色检测嗜碱性细胞形态和纯度。荧光分光光度法检测免疫球蛋白介导的组胺释放，定量RT-PCR检测IL-4和IL-13的产生。用流式细胞仪检测CD203c和CD63在抗IgE活化前后的表达。结果采用该方法后，大多数患者的嗜碱性粒细胞接近均一，平均纯度99.34+/-0.88%(范围97-100%，n=18)，平均回收率75.6(范围39-100%，n=8)。用台盼蓝拒染法纯化后的嗜碱性粒细胞存活率为99.6+/-0.89%。纯化程序使嗜碱性粒细胞对抗IgE的组胺释放以及IL-4和IL-13mRNA的表达具有正常的功能反应。结论该方法快速、简便、重复性好，有利于嗜碱性粒细胞在体外高产量研究中的应用。
Background Basophils are increasingly utilized as indicators of allergic inflammation and as primary allergic effector cells to study signalling pathways. However, until the present, their enrichment has been time consuming, costly and limited to relatively few specialized laboratories.Objective We have therefore devised a reproducible and rapid method for the purification of human basophils from small quantities of peripheral blood within 1.5 h, which does not require the use of specialized equipment such as elutriators.Methods Human basophils were obtained from healthy volunteers undergoing venipuncture. Heparinized or K3-ethylenediaminetetraacetic acid blood samples were first subjected to centrifugation in Hetasep, directly followed by negative selection using immunomagnetic beads. Basophil morphology and purity were assessed by May-Grunwald staining of cytospins. IgE-mediated histamine release was analysed spectrofluorometrically and IL-4 and IL-13 production by quantitative RT-PCR. CD203c and CD63 surface expression was measured using flow cytometry before and after activation with anti-IgE.Results Using this protocol, basophils were enriched close to homogeneity in most cases with a mean purity of 99.34 +/- 0.88% (range 97-100%, n = 18) and a mean recovery of 75.6 (range 39-100%, n = 8). Basophil viability following purification was 99.6 +/- 0.89% using Trypan blue exclusion. The purification procedure gave rise to basophils with normal functional responses to anti-IgE regarding histamine release as well as IL-4 and IL-13 mRNA expression. Moreover, constitutive cell-surface CD203c/CD63 expressions were not elevated before anti-IgE stimulation.Conclusion The rapidity, simplicity and reproducibility of this method will facilitate the employment of basophils in high-output ex vivo studies.