Olfactory receptors for isolvaleric acid and related malodorants and use thereof in assays for identification of blockers
a technology of isolvaleric acid and receptors, which is applied in the field of olfactory receptors for isolvaleric acid and related malodorants and assays for identification of blockers, can solve the problems of psychophysical testing of human subjects, application failure to provide the specificity of many of these receptors, and inability to identify whether, so as to block, inhibit, modulate, enhance the activation of isovaleric acid receptors
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example 1
Isolation and Imaging of Individual Murine Olfactory Neurons
[0214]In this example, the details of how the individual murine olfactory neurons were isolated from murine olfactory epithelium and subjected to Ca2+ imaging are described.
[0215]Olfactory neuroepithelium was dissected from Balb / C mice and placed in a small Petri dish with phosphate-buffered saline (PBS) without Ca2+ and Mg2+. The tissue was fragmented under the dissecting microscope into pieces as small as possible. The buffer was removed and replaced by 2 ml of fresh PBS without Ca2+ and Mg2+ supplemented with 0.025% trypsin, 0.75 mM EDTA for 15 min. After dissociation, the tissue was transferred into 5 ml PBS without Ca2+ and Mg2+ and the tissue was triturated very gently by pipetting up and down 4-5 times with plastic disposable, progress of cell dissociation was monitored under inverted microscope. After dissociation, the tissue was transferred into 10 ml of Dulbecco's modified Eagle medium (DMEM) supplemented with 10%...
example 2
Single-Cell cDNA Synthesis and PCR Amplification of or cDNA Fragments
[0218]The single neurons collected in lysis buffer were heated to 65° C. in a water for 1 min to lyse the cells, then kept at room temperature for 1-2 min to allow the oli primer to anneal to the RNA and put back on ice. The tube was centrifuged briefly μl of a 1:1 mix of AMV- and MMLV-reverse transcriptases (Gibco-BRL) were added followed by incubation for 15 min at 37° C. For control experiments, the reaction mi was divided into two equal aliquots—one for reverse transcriptase reaction (cDNA synthesis) and another for a negative control without reverse transcriptase. After incubation, the reverse transcriptases were inactivated by incubation for 10 min at 6 the tubes were put back on ice, then briefly centrifuged (2 min at 4° C.). 4.5 μl of 2× buffer (stock tailing buffer: 800 μl of 5× Gibco-BRL terminal transferase buffer, 30 μl 100 mM dATP, 1.17 ml H2O) containing 10 units of terminal transferase were added rea...
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