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A novel method to purify neutrophils enables functional analysis of zebrafish hematopoiesis
Genes to Cells ( IF 1.3 ) Pub Date : 2020-10-02 , DOI: 10.1111/gtc.12810
Katsuhiro Konno 1, 2 , Kasem Kulkeaw 3 , Manabu Sasada 4 , Takenobu Nii 1 , Ayako Kaneyuki 1 , Tohru Ishitani 5, 6 , Fumio Arai 2 , Daisuke Sugiyama 1, 7, 8
Affiliation  

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‐E4low DRAQ5low cells, 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‐Elow DRAQ5low populations. These cells had myeloperoxidase activity, were Sudan Black B‐positive and expressed high levels of neutrophil‐specific (csf3r and mpx) 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.

中文翻译:

纯化嗜中性粒细胞的新方法可以对斑马鱼的造血功能进行功能分析,纯化嗜中性粒细胞的新方法可以对斑马鱼的造血功能进行功能分析

斑马鱼是研究脊椎动物造血作用的有用模型,但是缺乏针对斑马鱼蛋白的抗体限制了造血细胞的纯化。在这里,我们使用凝集素菜豆红细胞凝集素(PHA-E)和DNA染色荧光染料DRAQ5纯化了幼虫和成年斑马鱼的嗜中性粒细胞。在成年肾中,我们纯化了嗜中性粒细胞样颗粒的中性粒细胞样PHA-E4DRAQ5细胞。具体而言,在受精后96小时,我们使用正向散射对幼虫的大型细胞进行了分类,发现它们由PHA-EDRAQ5种群组成。这些细胞具有髓过氧化物酶活性,苏丹黑B阳性,并表达高水平的中性粒细胞特异性(csf3rmpx)mRNA,所有中性粒细胞的特征。使用这种方法,我们进行了功能分析,表明zyxin(Zyx)在斑马鱼幼虫的中性粒细胞生成中起作用。总体而言,基于PHA-E和DRAQ5的流式细胞仪可作为纯化斑马鱼中性粒细胞的工具。,斑马鱼是研究脊椎动物造血作用的有用模型,但是缺乏针对斑马鱼蛋白的抗体限制了造血细胞的纯化。在这里,我们使用凝集素菜豆红细胞凝集素(PHA-E)和DNA染色荧光染料DRAQ5纯化了幼虫和成年斑马鱼的嗜中性粒细胞。在成年肾中,我们纯化了嗜中性粒细胞样颗粒的中性粒细胞样PHA-E4DRAQ5细胞。具体而言,在受精后96小时,我们使用正向散射对幼虫的大型细胞进行了分类,发现它们由PHA-EDRAQ5种群组成。这些细胞具有髓过氧化物酶活性,苏丹黑B阳性,并表达高水平的中性粒细胞特异性(csf3rmpx)mRNA,所有中性粒细胞的特征。使用这种方法,我们进行了功能分析,表明zyxin(Zyx)在斑马鱼幼虫的中性粒细胞生成中起作用。总体而言,基于PHA-E和DRAQ5的流式细胞仪可作为纯化斑马鱼中性粒细胞的工具。
更新日期:2020-12-11
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