A novel method to purify neutrophils enables functional analysis of zebrafish hematopoiesis
A novel method to purify neutrophils enables functional analysis of zebrafish hematopoiesis
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一种纯化中性粒细胞的新方法可以对斑马鱼造血功能进行功能分析
DOI：
10.1111/gtc.12810
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发表时间：
2020
期刊：
影响因子：
2.1
通讯作者：
Sugiyama Daisuke
中科院分区：
文献类型：
--
作者：
Konno Katsuhiro;Kulkeaw Kasem;Sasada Manabu;Nii Takenobu;Kaneyuki Ayako;Ishitani Tohru;Arai Fumio;Sugiyama Daisuke
斑马鱼是研究脊椎动物造血的有效模型，但缺乏对斑马鱼蛋白的抗体限制了造血细胞的纯化。在这里，我们使用凝集素Phaseolus vulgaris红凝集素（PHA‐E）和DRAQ5（一种DNA染色荧光染料）从斑马鱼幼虫和成年斑马鱼中纯化中性粒细胞。在成人肾骨髓中，我们纯化了嗜中性粒细胞样PHA - e4lowdraq5lowcell，其中嗜中性粒细胞型颗粒。具体来说，在受精后96小时，我们使用前向散射法从幼虫中分出大细胞，发现它们由PHA - elowdraq5low种群组成。这些细胞具有髓过氧化物酶活性，苏丹黑B阳性，表达高水平的中性粒细胞特异性（csf3randmpx） mrna，所有中性粒细胞特征。利用这种方法，我们进行了功能分析，表明zyxin （Zyx）在斑马鱼幼体中性粒细胞的产生中起作用。总的来说，基于PHA - E和DRAQ5的流式细胞术可以作为纯化斑马鱼中性粒细胞的工具。
Zebrafish is a useful model to study vertebrate hematopoiesis, but lack of antibodies to zebrafish proteins has limited purification of hematopoietic cells. Here, we purified neutrophils from larval and adult zebrafish using the lectin Phaseolus vulgaris erythroagglutinin (PHA‐E) and DRAQ5, a DNA‐staining fluorescent dye. In adult kidney marrow, we purified neutrophil‐like PHA‐E4lowDRAQ5lowcells, which neutrophil‐type granules. Specifically, at 96‐hr post‐fertilization, we sorted large‐sized cells from larvae using forward scatter and found that they consisted of PHA‐ElowDRAQ5lowpopulations. These cells had myeloperoxidase activity, were Sudan Black B‐positive and expressed high levels of neutrophil‐specific (csf3randmpx) mRNAs, all neutrophil characteristics. Using this method, we conducted functional analysis suggesting that zyxin (Zyx) plays a role in neutrophil generation in zebrafish larvae. Overall, PHA‐E and DRAQ5‐based flow cytometry serves as a tool to purify zebrafish neutrophils.