Search

The advent of soft lithography allowed for an unprecedented expansion in the field of microfluidics. However, the vast majority of PDMS microfluidic devices are still made with extensive manual labor, are tethered to bulky control systems, and have cumbersome user interfaces, which all render commercialization difficult. On the other hand, 3D printing has begun to embrace the range of sizes and materials that appeal to the developers of microfluidic devices. Prior to fabrication, a design is digitally built as a detailed 3D CAD file. The design can be assembled in modules by remotely collaborating teams, and its mechanical and fluidic behavior can be simulated using finite-element modeling. As structures are created by adding materials without the need for etching or dissolution, processing is environmentally friendly and economically efficient. We predict that in the next few years, 3D printing will replace most PDMS and plastic molding techniques in academia.

The advent of soft lithography allowed for an unprecedented expansion in the field of microfluidics. However, the vast majority of PDMS microfluidic devices are still made with extensive manual labor, are tethered to bulky control systems, and have cumbersome user interfaces, which all render commercialization difficult. On the other hand, 3D printing has begun to embrace the range of sizes and materials that appeal to the developers of microfluidic devices. Prior to fabrication, a design is digitally built as a detailed 3D CAD file. The design can be assembled in modules by remotely collaborating teams, and its mechanical and fluidic behavior can be simulated using finite-element modeling. As structures are created by adding materials without the need for etching or dissolution, processing is environmentally friendly and economically efficient. We predict that in the next few years, 3D printing will replace most PDMS and plastic molding techniques in academia.

We studied modulation of current in human embryonic kidney tsA-201 cells coexpressing rat erg1 channels with M1 muscarinic receptors. Maximal current was inhibited 30% during muscarinic receptor stimulation, with a small positive shift of the midpoint of activation. Inhibition was attenuated by coexpression of the regulator of G-protein signalling RGS2 or of a dominant-negative protein, Gq, but not by N-ethylmaleimide or C3 toxin. Overexpression of a constitutively active form of Gq (but not of G13 or of Gs) abolished the erg current. Hence it is likely that Gq/11, and not Gi/o or G13, mediates muscarinic inhibition. Muscarinic suppression of erg was attenuated by chelating intracellular Ca2+ to < 1 nm free Ca2+ with 20 mm BAPTA in the pipette, but suppression was normal if internal Ca2+ was strongly clamped to a 129 nm free Ca2+ level with a BAPTA buffer and this was combined with numerous other measures to prevent intracellular Ca2+ transients (pentosan polysulphate, preincubation with thapsigargin, and removal of extracellular Ca2+). Hence a minimum amount of Ca2+ was necessary for the inhibition, but a Ca2+ elevation was not. The ATP analogue AMP-PCP did not prevent inhibition. The protein kinase C (PKC) blockers staurosporine and bisindolylmaleimide I did not prevent inhibition, and the PKC-activating phorbol ester PMA did not mimic it. Neither the tyrosine kinase inhibitor genistein nor the tyrosine phosphatase inhibitor dephostatin prevented inhibition by oxotremorine-M. Hence protein kinases are not needed. Experiments with a high concentration of wortmannin were consistent with recovery being partially dependent on PIP2 resynthesis. Wortmannin did not prevent muscarinic inhibition. Our studies of muscarinic inhibition of erg current suggest a role for phospholipase C, but not the classical downstream messengers, such as PKC or a calcium transient.