Single-Cell Microarray for Analyzing Cellular Response

Abstract

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.

Identification of antigen‐specific B cells by concurrent monitoring of intracellular Ca2+ mobilization and antigen binding with microwell array chip system equipped with a CCD imager

“…Cytomics is becoming increasingly important for analyzing cell conditions and cellular responses to stimuli from extracellular milieu, which will contribute to drug discovery as well as predictive medicine. Cell microarray-based analysis offers one way for analyzing cytomics; it has been conventionally performed with a single parameter (11)(12)(13)(14)(15)(16)(17)(18). Here, for the first time, we present a method of two-parameter cell microarray-based time-lapse analysis.…”

“…To date, some cell microarray technologies using microwells have been reported for analyzing a large number of nonadherent cells (11)(12)(13)(14)(15)(16). These microwells are designed to capture single cells, enabling the tracking and analyzing of cellular functions at the single-cell level.…”

Single lymphocyte analysis with a microwell array chip

“…The splenocytes were prepared and loaded with 0.1 lM CellTracker orange (Ex, 535 nm; Em, 585 nm; Invitrogen, Carlsbad, CA) and 0.1 lM Fluo-4 (Ca 21 -dependent fluorophore; Ex, 473 nm; Em, 532 nm; Invitrogen), as previously described (6). The procedure for the transgenic mice experiments was approved by the Committee for Recombinant DNA Experiments (#19 -9) and Animal Experiments at the University of Toyama (# 2006-Med-33).…”

“…Cell microarray analysis was performed as previously described (6). Briefly, cells were loaded onto a microwell array chip and the Fluo-4 fluorescence of the individual cells before stimulation was measured by scanning the chip with a cell scanner (CRBIO IIe-FITC, Hitachi Software, Tokyo, Japan) that was modified from a DNA chip scanner (CRBIO IIe) by changing a 635-nm laser to a 473-nm laser and whose minimum resolution was improved to 2.5 lm.…”

“…To meet the demand for this type of analysis, we have developed an analysis system using a microwell array chip that has a large number of microwells whose size and shape just fit a single cell (6). By applying an individual cell to each microwell, an array of live cells was prepared, and the cellular responses of individual cells, such as alteration of intracellular Ca 21 concentration, were monitored using a fluorescent scanner that was modified to scan cells.…”