使用计算机控制微量移液器的单细胞黏附测定法。
Single cell adhesion assay using computer controlled micropipette.
作者信息
Salánki Rita, Hős Csaba, Orgovan Norbert, Péter Beatrix, Sándor Noémi, Bajtay Zsuzsa, Erdei Anna, Horvath Robert, Szabó Bálint
机构信息
Doctoral School of Molecular- and Nanotechnologies, University of Pannonia, Veszprém, Hungary; Nanobiosensorics Group, Research Centre for Natural Sciences, Institute for Technical Physics and Materials Science, Budapest, Hungary; Department of Biological Physics, Eötvös University, Budapest, Hungary.
Department of Hydrodynamic Systems, Budapest University of Technology and Economics, Budapest, Hungary.
出版信息
PLoS One. 2014 Oct 24;9(10):e111450. doi: 10.1371/journal.pone.0111450. eCollection 2014.
Cell adhesion is a fundamental phenomenon vital for all multicellular organisms. Recognition of and adhesion to specific macromolecules is a crucial task of leukocytes to initiate the immune response. To gain statistically reliable information of cell adhesion, large numbers of cells should be measured. However, direct measurement of the adhesion force of single cells is still challenging and today's techniques typically have an extremely low throughput (5-10 cells per day). Here, we introduce a computer controlled micropipette mounted onto a normal inverted microscope for probing single cell interactions with specific macromolecules. We calculated the estimated hydrodynamic lifting force acting on target cells by the numerical simulation of the flow at the micropipette tip. The adhesion force of surface attached cells could be accurately probed by repeating the pick-up process with increasing vacuum applied in the pipette positioned above the cell under investigation. Using the introduced methodology hundreds of cells adhered to specific macromolecules were measured one by one in a relatively short period of time (∼30 min). We blocked nonspecific cell adhesion by the protein non-adhesive PLL-g-PEG polymer. We found that human primary monocytes are less adherent to fibrinogen than their in vitro differentiated descendants: macrophages and dendritic cells, the latter producing the highest average adhesion force. Validation of the here introduced method was achieved by the hydrostatic step-pressure micropipette manipulation technique. Additionally the result was reinforced in standard microfluidic shear stress channels. Nevertheless, automated micropipette gave higher sensitivity and less side-effect than the shear stress channel. Using our technique, the probed single cells can be easily picked up and further investigated by other techniques; a definite advantage of the computer controlled micropipette. Our experiments revealed the existence of a sub-population of strongly fibrinogen adherent cells appearing in macrophages and highly represented in dendritic cells, but not observed in monocytes.
细胞黏附是所有多细胞生物至关重要的基本现象。识别并黏附特定大分子是白细胞启动免疫反应的关键任务。为了获得关于细胞黏附的具有统计学可靠性的信息,需要对大量细胞进行测量。然而,直接测量单个细胞的黏附力仍然具有挑战性,并且当今的技术通常通量极低(每天5 - 10个细胞)。在此,我们介绍一种安装在普通倒置显微镜上的计算机控制微量移液器,用于探测单个细胞与特定大分子的相互作用。我们通过对微量移液器尖端流动的数值模拟计算了作用于靶细胞的估计流体动力提升力。通过在位于被研究细胞上方的移液器中施加逐渐增加的真空来重复吸取过程,可以准确探测表面附着细胞的黏附力。使用所介绍的方法,在相对较短的时间内(约30分钟)逐个测量了数百个黏附于特定大分子的细胞。我们用蛋白质非黏附性的PLL - g - PEG聚合物阻断非特异性细胞黏附。我们发现人类原代单核细胞比其体外分化后代(巨噬细胞和树突状细胞)对纤维蛋白原的黏附性更低,后者产生的平均黏附力最高。通过静水阶跃压力微量移液器操作技术对本文介绍的方法进行了验证。此外,在标准微流控剪切应力通道中强化了该结果。然而,自动化微量移液器比剪切应力通道具有更高的灵敏度和更少的副作用。使用我们的技术,被探测的单个细胞可以很容易地被拾取并通过其他技术进一步研究;这是计算机控制微量移液器的一个明显优势。我们的实验揭示了在巨噬细胞中存在一个对纤维蛋白原有强黏附性的亚群细胞,在树突状细胞中高度富集,但在单核细胞中未观察到。
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