Capillary force-driven cell perfusion microchamber: calcium transients in isolated smooth muscle cells.
We present a new design for a sub-microlitre chamber which enables perfusion of individual cells. No equipment is required for it to be operational, as the exchange of solutions in the chamber is driven solely by capillary forces. Its active volume consists of a 30-micron-thin layer between two coverslips (separated by a couple of spacers) and is adjustable down to 0.1 microl. This slimline design (1) guarantees that all cells are kept in one (focal) plane during recording/perfusion (thus minimizing movement artefacts when intracellular fluorescence is monitored); (2) facilitates fixing of cells to a coverslip (often even without the use of poly-L-lysine, especially when small cell clusters are used); and (3) makes it possible to perfuse individual cells on a specimen stage of an upright microscope even under high-power objectives with a short working distance, which is of potential use in field studies where the smaller size of an upright microscope (compared to the inverted one) can be advantageous. As an example, we present a chamber with an active volume of approx. 0.5 microl and perfusion rate high enough to enable complete exchange of solution within 250 ms in an area of 500 micron x 500 micron. Only 1 microl of perfusing solution is required for exchanging the entire volume of the chamber. We present an example of intracellular free calcium transients in isolated smooth muscle cells upon release of intracellular sequestered calcium.
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University Laboratory of Physiology, Parks Road, Oxford OX1 3PT, UK.
Ashley
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D000814:Aniline Compounds; D000818:Animals; D049872:Bivalvia / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism*; D002197:Capillary Action; D005456:Fluorescent Dyes; D009130:Muscle, Smooth / Q000378:metabolism*; D010477:Perfusion; D012996:Solutions; D013050:Spectrometry, Fluorescence; D014966:Xanthenes
D000814:Aniline Compounds; D005456:Fluorescent Dyes; D012996:Solutions; D014966:Xanthenes; C059715:Fluo-3; D002118:Calcium