Departmental Affiliation and City, State, Zip for All Authors

Department of Anatomy and Physiology UNT Health Science Center, Fort Worth, Texas

Scientific Abstract

Traditionally, it has been difficult to detect the release of neuropeptides, such as Angiotensin II (ANG II), with a temporal and spatial resolution that is consistent with the time course of neural function and the size of neurons. We employed the use of sniffer cells as a means to detect neuropeptide release. Sniffer cells are Chinese Hamster Ovary (CHO) cells that have been transfected with plasmids to express angiotensin type 1a (AT1a) receptor, as well as a genetically encoded calcium sensor (RGECO or GCAMP). Transfected cells were placed into a fluorescent activated cell sorting (FACS) machine and individually sorted into a 96-well plate based on fluorescent intensity. The individual cells were left to proliferate to create single cell clonal lines. Sniffer cells were plated onto glass cover slips and continuously perfused with artificial cerebral spinal fluid (aCSF). We measured the fluorescent intensity of the cells in response to 1 minute perfusion of various compounds. In preliminary experiments, the sniffer cells showed a fluorescent increase in response to ANG II and ANG III, but was unresponsive to other common neurotransmitters. These cells were shown to have a dose-dependent response to the ANG II and ANG III but had great variability. Use of FACS on sniffer cells minimized variability in gene expression resulting in more consistent detection and quantification of neuropeptide release in neurons. The sniffer cell technique has shown to be a viable method to detect neuropeptides on a rapid time course.

Share

COinS
 

Faster Than Ever: Rapid Detection of Neuropeptide Release Using "Sniffer Cells"

Traditionally, it has been difficult to detect the release of neuropeptides, such as Angiotensin II (ANG II), with a temporal and spatial resolution that is consistent with the time course of neural function and the size of neurons. We employed the use of sniffer cells as a means to detect neuropeptide release. Sniffer cells are Chinese Hamster Ovary (CHO) cells that have been transfected with plasmids to express angiotensin type 1a (AT1a) receptor, as well as a genetically encoded calcium sensor (RGECO or GCAMP). Transfected cells were placed into a fluorescent activated cell sorting (FACS) machine and individually sorted into a 96-well plate based on fluorescent intensity. The individual cells were left to proliferate to create single cell clonal lines. Sniffer cells were plated onto glass cover slips and continuously perfused with artificial cerebral spinal fluid (aCSF). We measured the fluorescent intensity of the cells in response to 1 minute perfusion of various compounds. In preliminary experiments, the sniffer cells showed a fluorescent increase in response to ANG II and ANG III, but was unresponsive to other common neurotransmitters. These cells were shown to have a dose-dependent response to the ANG II and ANG III but had great variability. Use of FACS on sniffer cells minimized variability in gene expression resulting in more consistent detection and quantification of neuropeptide release in neurons. The sniffer cell technique has shown to be a viable method to detect neuropeptides on a rapid time course.

Manuscript Number

1052