Masahiro Omori | PubMed Health

PubMed2005

Single-cell microarray for analyzing cellular response.

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.

Amino Acid Sequence↗

PubMed2005

Formaldehyde-limited cultivation of a newly isolated methylotrophic bacterium, Methylobacterium sp. MF1: enzymatic analysis related to C1 metabolism.

Formaldehyde is a highly toxic compound to most living organisms. We have isolated a bacterial strain that is able to efficiently degrade formaldehyde and use it as a sole carbon source. The isolated strain was identified as Methylobacterium sp. MF1, which could grow on formaldehyde and methanol. Methylobacterium sp. MF1 was grown in batch culture using 1.2 g/l formaldehyde as a sole carbon source, which was all consumed within 200 h. In order to decompose formaldehyde more efficiently, formaldehyde-limited chemostat cultivation of Methylobacterium sp. MF1 was investigated. Formaldehyde was consumed at 1.7 g/l/d when the dilution rate was 0.012 h(-1). Under these conditions, the cell turbidity (OD610) reached 2.0. Furthermore, when the initial turbidity was adjusted to 3.0 using methanol-grown cells, continuous cultivation could be started at an initial dilution rate of 0.008 h(-1). Using these conditions, consumption of formaldehyde could be continued for at least 600 h. The enzyme activities of cells growing as a chemostat culture, using methanol or formaldehyde as a sole carbon source, were compared to that of C1 metabolism. No difference was detected in the enzyme activities for the oxidation and assimilation of C1 compounds between the two cell-free extracts. Furthermore, methanol dehydrogenase activity was detected at the same level when formaldehyde was used as a sole carbon source. These results suggest that the resistance to the toxic effects of formaldehyde exhibited by Methylobacterium sp. MF1 is related to factors other than C1 metabolism.

Aldehyde Oxidoreductases↗