Navigation Links
The use of CypHer5E and the IN Cell Analyzer 1000 for live-cell receptor internalization studies

>
Materials


Other materials required
HEK293 cell line expressing VSV-G epitope tagged β2-adrenergic receptor

HEK293 cell line expressing VSV-G epitope tagged δ-opioid receptor

HEK293 cell line expressing EGF receptor

HEK Culture medium: MEM (Sigma M2279) containing:
10% (v/v) FBS
Geneticin G418, 200 µg/ml (Sigma)
L-glutamine (Gibco)
Non-essential amino acids (Sigma)

Assay medium: MEM, phenol red free (Invitrogen 51200-038), or Krebs-Ringer buffer (see method)

Agonists: Isoproterenol, DADLE ([D-Ala2, D-Leu5]-Enkephalin), EGF (epidermal growth factor, murine submaxillary gland) (Sigma)

Hoechst 33342 (Molecular Probes)

Rat anti-human EGFR (Serotec, MCA1784)

Phosphate-buffered saline (PBS) solution

Poly-D-lysine, 5 mg (Sigma P6407)

96-well assay plate (Packard Viewplate)


Methods
Assay for β2-adrenergic and δ-opioid receptor internalization

1. Pre-coat 96-well plates with poly-D-lysine, recommended for HEK293 cell lines.

2. Seed cells at a density of ~ 16 000 cells/well.

3. Incubate ~ 48 h at 37 °C with 5% CO2.

4. Replace the culture medium with 100 µl/well of fresh MEM (phenol red free) containing 2.5–5 µg/ml CypHer5E anti-VSV-G antibody and 2.5 µM Hoechst.

5. Incubate at room temperature for 15 min.

6. Add 50 µl assay medium containing agonist and incubate at 37 °C for 30 min.

7. Image on the IN Cell Analyzer 1000 (20× objective), exposure time 300 ms/field, 620 nm excitation and 700 nm emission for CypHer5E; exposure time o
'"/>

Source:


Page: All 1 2 3 4 5 6

Related biology technology :

1. Screening for potential beta 2-adrenergic receptor antagonists using CypHer5E and IN Cell Analyzer 1000
2. Analysis of in vitro micronucleus assays using the IN Cell Analyzer 3000 Micronuclei Formation Analysis Module
3. Screening for β2-adrenergic receptor agonists using the pH-sensitive dye,CypHer5, and the IN Cell Analyzer 3000
4. Identification of cells in S phase using the Cell Proliferation Fluorescence Kit and IN Cell Analyzer 1000
5. Rapid, High Throughput Mutation Discovery Using Fluorescent SSCP on the ABI PRISM 3100 and 3100Avant Genetic Analyzers
6. The development of a CXCR2 chemokine receptorbinding assay using the LEADseeker Multimodality Imaging System
7. The development of a melanocortin MC4 receptor binding assay using the LEADseeker Multimodality Imaging System
8. The development of a melanocortin MC5 receptor binding assay using the LEADseeker Multimodality Imaging System
9. The development of a calcitonin-gene related peptide (CGRP) receptor-binding assay using the LEADseeker Multimodality Imaging System
10. The use of CypHer5 for receptor internalization studies in both a range of GPCRs and a non-GPCR
11. Development of a histamine H1 receptor binding assay using the LEADseeker Multimodality Imaging System
Post Your Comments:
(Date:4/23/2015)... Jose, California (PRWEB) April 23, 2015 ... Predictive diagnostics refer to a series of genetic ... as cystic fibrosis, Lou Gehrig's disease, Huntington's disease, ... in examining the gene composition and interaction in ... the disease as well as the response to ...
(Date:4/23/2015)... Proove Biosciences , a commercial ... to announce the launch of Proove Non-Opioid ... 62 genetic variations to predict good versus poor ... non-opioid pain medications, namely ibuprofen (brand name Advil, ... Xanax) and acetaminophen (brand name Tylenol). , ...
(Date:4/23/2015)... CA (PRWEB) April 23, 2015 Delpor, ... today announced that the United States Patent and Trademark ... Company’s NANOPOR™ technology. US Pat. No. ... Delpor’s microfabricated drug delivery device for the ... proprietary NANOPOR™ technology for the sustained (zero-order) release of ...
(Date:4/22/2015)... Today, DuPont announced it has ... microbiome discovery company. This acquisition will build on ... both seed and crop protection to discover and ... , “In 2014, the businesses in DuPont’s Agriculture, ... more than $1 billion in biological solutions across ...
Breaking Biology Technology:Increasing Disease Prevalence & Focus on Preventive Healthcare Drive the Predictive Diagnostics Market, According to a New Report by Global Industry Analysts, Inc. 2Increasing Disease Prevalence & Focus on Preventive Healthcare Drive the Predictive Diagnostics Market, According to a New Report by Global Industry Analysts, Inc. 3Proove Biosciences Launches World’s First Test to Predict Responders to Commonly Prescribed Non-Opioid Pain Medications 2Proove Biosciences Launches World’s First Test to Predict Responders to Commonly Prescribed Non-Opioid Pain Medications 3Delpor, Inc. Announces the Issuance of 2 New US Patents Based on its Nanopore Technology which Enables the Sustained Release of Drugs from a Small Implant Device 2Delpor, Inc. Announces the Issuance of 2 New US Patents Based on its Nanopore Technology which Enables the Sustained Release of Drugs from a Small Implant Device 3DuPont Acquires Taxon Biosciences, Inc. 2DuPont Acquires Taxon Biosciences, Inc. 3DuPont Acquires Taxon Biosciences, Inc. 4
... Draper Laboratory, in conjunction with the National Academy of Engineering (NAE), ... about the impact engineers have had on our daily lives, and ... awarded "Engineering,s Nobel" since 1989. , ... Cambridge, MA ...
... Inc. (Nasdaq: SVNT ) today announced the ... Vice President, Chief Financial Officer & Treasurer. Mr. ... Savient,s Chief Financial Officer, effective immediately. "We are ... Vice President of Corporate Finance and Controller, David will ...
... SAN DIEGO, Feb. 12 Volcano Corporation,(Nasdaq: ... sales of,products for the diagnosis and treatment of coronary ... participating in the Roth Capital Partners,21st Annual Growth Stock ... The presentation by Scott Huennekens, president and chief ...
Cached Biology Technology:New Educational Website Celebrates the Accomplishments of Engineers 2Savient Announces Appointment of David Gionco as Chief Financial Officer 2Savient Announces Appointment of David Gionco as Chief Financial Officer 3Savient Announces Appointment of David Gionco as Chief Financial Officer 4Volcano Corporation Presentation at Roth Capital Partners Conference to Be Webcast 2
(Date:3/24/2015)... , Mar. 24, 2015 Research and Markets ... of the "Global Iris Recognition Market 2015-2019" ... the Global Iris Recognition market to grow at a ... This report covers the present scenario and the ... the period 2015-2019. To calculate the market size, the ...
(Date:3/24/2015)... 24, 2015   Crossmatch ™, a leading ... that its U.are.U ® 4500 Fingerprint Readers ... Bros . to achieve PCI compliance for Point-of-Sale ... to the POS terminal, protecting the retailer and ... breaches. With one-third of worldwide ...
(Date:3/23/2015)... , March 23, 2015  In the 2014 fiscal ... case conviction losses. This figure accounts for a fraction ... to The Nilson Report, exceeds $11 billion globally. The ... after a crime has occurred. Investor Mark Saint ... expert Joe Paolella partnered to administer the ...
Breaking Biology News(10 mins):Global Iris Recognition Market 2015-2019 with 3M Cogent, Bioenable Technologies, Cross Match Technology, Iris ID Systems & Safran Dominating 2Crossmatch Fingerprint Readers Help Retailer Prevent Data Breaches at Point-of-Sale 2Polygraph Test Now Used to Protect Investors From Fraud 2Polygraph Test Now Used to Protect Investors From Fraud 3
... - A Purdue University study shows dairy has an advantage ... Connie Weaver, distinguished professor and head of the food and ... dry milk were longer, wider, more dense and stronger than ... carbonate is the most common form of calcium used in ...
... biologist Dr. Gary G. Borisy, director and CEO of ... The National Academy of Sciences (NAS), an honorific society ... The achievement, considered one of the highest honors in ... Academy,s 146th annual meeting in Washington, D.C. Dr. Borisy ...
... calve from the northern front of the Wilkins Ice Shelf ... follows the collapse three weeks ago of the ice bridge ... The ice bridge, which effectively formed a barrier pinning ... Shelf, collapsed on 5 April removing about 330 sq km ...
Cached Biology News:Purdue study finds dairy better for bones than calcium carbonate 2MBL director and CEO and cell biology pioneer Gary Borisy elected to National Academy of Sciences 2MBL director and CEO and cell biology pioneer Gary Borisy elected to National Academy of Sciences 3Satellite imagery shows fragile Wilkins Ice Shelf destabilized 2
Recommended for use as a cell culture substratum at 0.1-0.2 mg/cm 2 or 5-10 μl/cm 2 . Optimal concentration depends on cell type as well as the application or research objectives. Type B...
... The Eclipse is a state-of-the-art separation ... Fractionation (AFFF) technique. The Eclipse integrates easily ... DAWN, miniDAWN Tristar, as well as the ... size and molar mass distributions of the ...
... for determining the absolute count of CD34+ ... peripheral blood samples, and leukapheresis samples. These ... cell count and a total CD45 (leucocyte) ... use in the BD Procount progenitor cell ...
... Protein A and, until recently, no readily available ... purification. Standard methods for IgM purification generally are ... for removing all of the major impurities present ... Pierce has developed an easy-to-use affinity matrix and ...
Biology Products: