AERIUS® IMAGER
Applications for the Aerius Imaging System
IN-CELL WESTERN ASSAY
Application Overview
What is the In-Cell Western™ (ICW)?
A quantitative immunofluorescent assay for simultaneous analysis of two different proteins in fixed cultured cells.
[ABOVE] graphic courtesy of Paul Jasper, Harvard
[ABOVE] Kinetics of cellular ß-catenin accumulation upon stimulation with Wnt3a. (A) Time- and dose-dependent accumulation of cellular ß-catenin. L-cells were incubated with Wnt3a, then stained for ß-catenin (yellow in merged image) and DNA content (red). Plates were imaged with the Odyssey. (B) Quantification of ß-catenin accumulation. Levels were upregulated within 30 min of Wnt3a stimulation, exhibit increased intensity between 6–8 h, and start to plateau after 10 h. Graph shows two independent experiments, each done in quadruplicate.
Reprinted with permission from Hannoush, RN. PLoS One. 3(10):e3498 (2008).
With the ICW, you can:
Detect your target proteins in fixed cells, using target-specific primary antibodies and infrared-labeled secondary antibodies
Quantify total fluorescent signal from cell population
in each wellPerform ratiometric analysis with two distinct infrared fluorescent channels
Perform the assay in 96- or 384-well plates
Enhance the accuracy and biological relevance of your data by detecting proteins in situ
Advantages
In-Cell Western™ assays provide many unique advantages:
Sensitive, quantitative analysis of cellular signaling pathways
Reduced autofluorescent interference from cells, microplates,
and chemical compounds
“Snapshot” of signal transduction or protein expression of cell population in each well
Enhanced throughput
Eliminate cell lysis, gel loading, electrophoresis, and membrane transfer
Streamline the experimental procedure and data analysis to easily process
many samples at once
Increased precision
Use ratiometric analysis to normalize for variations in cell number
Quantify small changes in protein levels reliably
Easily screen cell treatments or drug candidates for their effects on target proteins
Obtain more relevant results than enzyme assays with purified proteins
[ABOVE] Time course of caspase-3 activation in S2 cells. (A-C) In-Cell Western analysis of S2 cells treated with Actinomycin D (Act D) to induce apoptosis. Each time point was measured in triplicate and stained for anti-active-caspase-3 (A; green) and f-actin (B; red, stained with near-infrared fluorescent phalloidin). Panel C shows merged pseudocolor images. (D) Active-caspase-3 protein levels from (A) were quantified and normalized to f-actin levels in (B) for each time point. The active caspase-3:f-actin ratio at 0min Actinomycin D exposure was designated as 1, and all other ratios are shown relative to this value. Error bars represent the standard error of each independent measurement. Exposure of S2 cells to Actinomycin D increased the relative levels of active caspase-3 over time.
Reprinted with permission from Bond, D.et al. Biol Proced Online. 10(1):20-28(2008).
Workflow
Techincal Resources
*For resources related to individual steps of the In-Cell Western assay, see the Workflow tab.
Protocols and Documents:
"Quantitative Methods for analysis of protein phosphorylation in drug development"
Olive, D.M. Expert Rev. Proteomics 1:327–341 (2004)"Quantitative Measurement of the Activation of Signaling Pathways Using Two-Color Infrared Fluorescent Western Blotting and Cell-Based Assays"
Chen, H. and D.M. Olive, poster presentation, IBC International Conference on Protein Kinases (2002)"A cell-based immunocytochemical assay for monitoring kinase signaling pathways and drug efficacy"
H. Chen, et al. Anal. Biochem. 338:136-142 (2005)For a complete list of pack inserts and protocols, click here
Webinars & Video Tutorials:
“The In-Cell Western™ Assay”
Dr Amy Schutz-Geschwender, LI-COR“Characterizing GPCR Function”
Dr Chris Kearn, University of Washington“In-Cell Western™ (ICW) Assay; Power User Guide”
Ashlee Muller, LI-COR“In-Cell Western™ Assay: From Cell Proliferation to Protein Trafficking"
Dr Harry Osterman, LI-COR
PubMed
Odyssey® Publications: In-Cell Western™ Assays 1-2007 – 6/2009
Odyssey Publications: RNAi interference 1/2007-6/2009
- Analysis of the kinetics of cell signaling and phosphorylation
Kumar et al.
Multipathway model enables prediction of kinase inhibitor cross-talk effects on migration of Her2-overexpressing mammary epithelial cells.
Mol Pharmacol 73:1668–1678 (2008)
The authors evaluated a method for predictive modeling of “off-target” and “pathway crosstalk” effects of small molecule kinase inhibitors. They used the ICW assay to perform quantitative, multipathway measurements of signaling events in a higher-throughput manner. With this method, they were able to predictively understand how kinase inhibitor treatment influences cell migration behavior.Hannoush, RN.
Kinetics of Wnt-driven β-catenin stabilization revealed by quantitative and temporal imaging.
PLoS ONE 3(10):e3498 (2008)
The author used the ICW assay as a high-throughput, quantitative method for measuring Wnt-induced changes in β-catenin protein levels. The assay revealed an unprecedented level of detail about the kinetics of β-catenin accumulation. The author suggests that the assay would be appropriate for secondary screening to quickly determine whether inhibitors of Wnt/β-catenin signaling act upstream or downstream of β-catenin.Chen, WW et al.,
Input–output behavior of ErbB signaling pathways as revealed by a mass action model trained against dynamic data.
Mol Syst Biol 5:239 (2009)Du, Y et al.
In-Cell Western analysis of Helicobacter pylori-induced phosphorylation of extracellular-signal related kinase via the transactivation of the epidermal growth factor receptor
Microbes Infect 9(7):838-46 (2007)
Du et al developed an ‘In-Cell Western’ (ICW) assay for quantitative examination of H. pylori-induced epithelial signalling to determine the role of the EGF receptor (EGFR) in H. pylori-induced phosphorylation of ERK in epithelial cells. Helicobacter pylori activates extracellular-signal related (ERK) kinases in gastric epithelial cells via transactivation of EGFR. H. pylori activation of EGFR may be relevant to e pithelial hyperproliferation and gastric carcinogenesis. H. pylori strains were co-incubated with A431 and AGS cells. pERK and total ERK were quantified in situ using ICW analysis. The EGFR inhibitor EKB-569 dose-dependently reduced H. pylori-induced ERK phoshorylation in A431 and AGS cells. The data suggested that H. pylori induces pERK in epithelial cells partly via the EGFR pathway. Additional signalling mechanisms are likely to be involved in H. pylori-induced ERK phosphorylation. ICW analysis is a rapid quantitative method for evaluating the effects of inhibitors on H. pylori-induced cell signalling pathways of relevance to gastric carcinogenesis.Wong, S.
A 384-well cell-based phosho-ERK assay for dopamine D2 and D3 receptors
Anal Biochem. 333(2): 265-72 (2004)
- Quantification of “off-target” drug effects in signaling pathway analysis
Kumar, N et al.
Multipathway model enables prediction of kinase inhibitor cross-talk effects on migration of Her2-overexpressing mammary epithelial cells.
Mol Pharmacol. 73(6):1668–78 (2008).
The authors evaluated a method for predictive modeling of “off-target” and “pathway crosstalk” effects of small molecule kinase inhibitors. They used the ICW assay to perform quantitative, multipathway measurements of signaling events in a higher-throughput manner. With this method, they were able to predictively understand how kinase inhibitor treatment influences cell migration behavior.
- Cell-based determinations of IC50 concentrations and efficacy testing of drug compounds
Maira SM et al.
Identification and characterization of NVP-BEZ235, a new orally available dual phosphatidylinositol 3-kinase/mammalian target of rapamycin inhibitor with potent in vivo antitumor activity.
Mol Cancer Ther. 7(7):1851-63 (2008)Yeh et al.
Biological characterization of ARRY-142886 (AZD6244), a potent, highly selective mitogen-activated protein kinase kinase 1/2 inhibitor.
Clin Cancer Res 13(5):1576-83 (2007)Chen et al.
A cell-based immunocytochemical assay for monitoring kinase signaling pathways and drug efficacy.
Analytical Biochemistry 338:136–142 (2005)
- Cell surface protein analysis and quantification
see On-Cell Western
- Receptor trafficking and recycling assays
see On-Cell Western
- Monitoring of GPCR activation
Selkirk, JV et al.
A novel cell-based assay for G-protein-coupled receptor-mediated cyclic adenosine monophosphate response element binding protein phosphorylation.
J Biomol Screen. 11(4): 351-8 (2006)
The authors validated the use of ICWs as a quantitative readout of GPCR function. Specifically, they used CREB phosphorylation as a downstream readout of GPCR activity in whole cells after treatment with receptor agonists. They validated this as a reliable high-throughput readout by comparing the results to traditional radioligand binding assays and cAMP competitive immunoassays.Wacker, JL et al.
Disease-causing mutation in GPR54 reveals the importance of the second intracellular loop for class A G-protein-coupled receptor function.
J Biol Chem. 283(45):31068-78 (2008)
- Cell proliferation and cell number assays
Widberg, CH et al.
Fibroblast growth factor receptor 1 is a key regulator of early adipogenic events in human preadipocytes.
Am J Physiol Endocrinol Metab. 296(1):E121-31 (2009).Montagut, C et al.
Elevated CRAF as a Potential Mechanism of Acquired Resistance to BRAF Inhibition in Melanoma.
Cancer Res. 68(12):4853-61 (2008)Kim, WJ et al. Blood.
Mutations in the neutral sphingomyelinase gene SMPD3 implicate the ceramide pathway in human leukemias.
111(9):4716-22 (2008).Godin-Heymann, N et al.
The T790M “gatekeeper” mutation in EGFR mediates resistance to low concentrations of an irreversible EGFR inhibitor.
Mol Cancer Ther. 7(4):874-9 (2008)Quinlan, MP et al.
Activated Kras, but Not Hras or Nras, May Initiate Tumors of Endodermal Origin via Stem Cell Expansion.
Mol Cell Biol. 28(8):2659-74 (2008)
- Surface protein expression on living cells
Delisle, BP et al.
Small GTPase determinants for the Golgi processing and plasmalemmal expression of human ether-a-go-go related (hERG) K+ channels.
J Biol Chem. 284(5):2844-53 (2009)
Delisle et al developed a ‘live-cell’ Western assay that quantitatively measures plasmalemmal expression of hERG by imaging living cells on tissue culture plates. This assay employs an antibody against an extracellular epitope in the hERG S1-S2 linker which is then detected by a IRDye 700 secondary antibody on Odyssey. These live-cell Western data recapitulates the findings from the Western blot, whole-cell patch clamp, and immunocytochemical analyses.
- Determination of viral titer and infectivity
Lin, YC et al.
Vaccinia virus DNA ligase recruits cellular topoisomerase II to sites of viral replication and assembly.
J Virol. 82: 5922-32 (2008)
The In-Cell Western assay was used to image viral plaque size and number in a study of the role of cellular topoisomerases in vaccinia virus replication and assembly.Sklan et al,
A Rab-GAP TBC Domain Protein Binds Hepatitis C Virus NS5A and Mediates Viral Replication.
J Virol. 81: 11096–11105 (2007)Counihan, NA et al.
Infrared fluorescent immunofocus assay (IR-FIFA) for the quantitation of non-cytopathic and minimally cytopathic viruses.
J Virol. 133:62-69 (2006)
