Technical advance: autofluorescence-based sorting: rapid and nonperturbing isolation of ultrapure neutrophils to determine cytokine production.
Technical advance: autofluorescence-based sorting: rapid and nonperturbing isolation of ultrapure neutrophils to determine cytokine production.
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DOI：
10.1189/jlb.0113040
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发表时间：
2013-07
影响因子：
5.5
通讯作者：
Rossi AG
中科院分区：
文献类型：
--
作者：
Dorward DA;Lucas CD;Alessandri AL;Marwick JA;Rossi F;Dransfield I;Haslett C;Dhaliwal K;Rossi AG
一种通过流式分选超纯化未受刺激的中性粒细胞并随后证明中性粒细胞在炎症刺激后释放IL-1β的能力的新方法。分离中性粒细胞而不污染白细胞，同时使中性粒细胞活化最小化的技术限制是确定特定中性粒细胞功能的障碍。我们的目的是评估使用流式细胞术在无抗体环境中产生高纯度的静止中性粒细胞群。通过不连续Percoll梯度分离外周血人粒细胞和小鼠骨髓来源的中性粒细胞，并使用FSC/SSC谱和自体荧光差异进行流式分选。通过形态学分析和流式细胞术评估分选后纯度。通过测量形状变化、CD 62 L和CD 11b表达、细胞内钙通量和趋化性，在未刺激的未分选和分选细胞中以及对fMLF、LTB 4和PAF的响应中测量神经元活化。还测定了人嗜中性粒细胞的细胞因子产生。分选后的人中性粒细胞纯度为99.95%（sem=0.03; n=11;形态学分析），99.68%为CD 16 +ve（sem=0.06; n=11），与鼠中性粒细胞获得的结果相似。流式细胞分选没有改变中性粒细胞的活化或趋化性，相对于预分选细胞，并没有观察到对激动剂的反应差异。刺激的嗜中性粒细胞产生IL-1β，尽管程度低于CXCL 8/IL-8。利用中性粒细胞和嗜酸性粒细胞之间的自体荧光的差异，通过流式细胞术是一种快速和有效的方法，为后续的体外研究产生高度纯化的群体。
A novel method to ultra-purify unstimulated neutrophils by flow sorting and subsequent demonstration of the neutrophils' ability to release IL-1β following inflammatory stimulus. The technical limitations of isolating neutrophils without contaminating leukocytes, while concurrently minimizing neutrophil activation, is a barrier to determining specific neutrophil functions. We aimed to assess the use of FACS for generating highly pure quiescent neutrophil populations in an antibody-free environment. Peripheral blood human granulocytes and murine bone marrow-derived neutrophils were isolated by discontinuous Percoll gradient and flow-sorted using FSC/SSC profiles and differences in autofluorescence. Postsort purity was assessed by morphological analysis and flow cytometry. Neutrophil activation was measured in unstimulated-unsorted and sorted cells and in response to fMLF, LTB4, and PAF by measuring shape change, CD62L, and CD11b expression; intracellular calcium flux; and chemotaxis. Cytokine production by human neutrophils was also determined. Postsort human neutrophil purity was 99.95% (sem=0.03; n=11; morphological analysis), and 99.68% were CD16+ve (sem=0.06; n=11), with similar results achieved for murine neutrophils. Flow sorting did not alter neutrophil activation or chemotaxis, relative to presorted cells, and no differences in response to agonists were observed. Stimulated neutrophils produced IL-1β, although to a lesser degree than CXCL8/IL-8. The exploitation of the difference in autofluorescence between neutrophils and eosinophils by FACS is a quick and effective method for generating highly purified populations for subsequent in vitro study.
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影响因子：
5.5
作者：
Boxio, R;Bossenmeyer-Pourié, C;Nüsse, O
通讯作者：
Nüsse, O
影响因子：
6
作者：
Cotter, MJ;Norman, KE;Ridger, VC
通讯作者：
Ridger, VC
影响因子：
3.2
作者：
Eversole, RR;Mackenzie, CD;Beuving, LJ
通讯作者：
Beuving, LJ
影响因子：
3.9
作者：
Sabroe, I;Prince, LR;Whyte, MKB
通讯作者：
Whyte, MKB
影响因子：
3.7
作者：
Alessandri AL;Duffin R;Leitch AE;Lucas CD;Sheldrake TA;Dorward DA;Hirani N;Pinho V;de Sousa LP;Teixeira MM;Lyons JF;Haslett C;Rossi AG
通讯作者：
Rossi AG