Single-cell microarray for analyzing cellular response
Single-cell microarray for analyzing cellular response
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DOI：
10.1021/ac0515632
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发表时间：
2005-12-15
影响因子：
7.4
通讯作者：
Muraguchi, A
中科院分区：
文献类型：
--
作者：
Yamamura, S;Kishi, H;Muraguchi, A
通过钙离子依赖的荧光染料测量细胞内钙离子(Ca~(2+))(I)来检测细胞反应是检测配体刺激的细胞和研究配体/受体相互作用信号的标准方法。我们描述了一种使用微室阵列芯片分析单个细胞如淋巴细胞的细胞反应的单细胞微阵列系统。单细胞微阵列芯片是由聚苯乙烯制成的，有超过3万个微室，只能容纳单个细胞。来自小鼠脾或人血的淋巴细胞在微阵列上铺展，80%以上的微室达到单细胞状态。通过芯片上的抗原受体刺激B细胞，我们可以通过将单个B细胞刺激前后的状态与抗原进行比较来检测激活的B细胞，这对于流式细胞仪是无效的。此外，这种新的方法还展示了利用显微操作器从微室中提取阳性单个B细胞，并实现了抗体DNA分析。该系统适用于单细胞水平的高通量细胞内钙离子反应分析，适用于抗原特异性淋巴细胞的筛选，用于制备特异性的单抗。
Detection of cellular response by measuring intracellular calcium, (Ca2+)(i), with Ca2+-dependent fluorescent dye are standard approaches to detect ligand-stimulated cells and to study signaling through ligand/receptor interaction. We describe a single-cell microarray system to analyze cellular response of individual cells such as lymphocytes using microchamber array chips. The single-cell microarray chip is made from polystyrene with over 30 000 microchambers, which can accommodate only single cells. Lymphocytes derived from mouse spleen or human blood were spread on the microarray, and over 80% of the microchambers achieved single-cell status. Stimulation of B-cells through antigen receptors on the microarray allowed us to detect activated B-cells by comparing the states of single B-cells before and after stimulation with antigen, which is disabled for flow cytometry. In addition, this novel method demonstrated retrieval of positive single B-cells from microchambers by a micromanipulator and achieved antibody DNA analysis. The system is suitable for high-throughput analysis of intracellular Ca2+ response at the single-cell level and is applicable to screen antigen-specific lymphocytes for making specific monoclonal antibody.