What’s all this BUZZZZ you are hearing about being able to quantitate cell signaling in plate-based assays? If you are at AACR in Chicago this week, stop by Booth 3800 (LI-COR® Biosciences) and we can tell you all about the In-Cell Western™ Assay – and how you can use this method to quantitate signaling, look at levels of protein phosphorylation, perform RNAi studies, monitor gene expression levels, conduct cell proliferation assays, and more. Imaging can be performed on the Odyssey® CLx, Odyssey Classic, or the Odyssey Sa Infrared Imager (the Sa also has the option for automation and barcode reading). And, if you can’t make it to AACR, stay tuned here and I will be blogging about this topic over the next week or so.
Okay, let’s start at the beginning. So what – exactly – is an In-Cell Western Assay? Well, some call it a cytoblot. To others, it’s a cell-based ELISA or an In-Cell ELISA (ICE Assay). To LI-COR, it’s a In-Cell Western Assay (we call it an ICW, for short) and is a quantitative immunofluorescence assay performed in microplates (96- or 384-well format). It combines the specificity of Western blotting with the reproducibility and throughput of ELISA.
In a nutshell, the basic steps are:
- Culture cells in microplates
- Treat cells
- Fix and permeabilize
- Stain with primary antibodies – 1 or 2 protein targets per well
- Stain with IRDye secondary antibody conjugates
- Image microplate and quantify fluorescent signals from cell populations in each well
- Quantify relative protein levels
- Normalize to correct for well-to-well variation
That doesn’t sound too difficult, right? Of course, just like any scientific technique, there are things to keep in mind to make sure your experiment gives the best, clearest, most accurate and reproducible results it can. In the next posts, I’ll share some of the technical tips to keep in mind – plus examples of how your research colleagues have used In-Cell ELISAs in their published papers.
In the meantime, here is a brochure on near-infrared applications for the Odyssey Imaging Systems, which includes a little more info on the technique and some examples with data. We also have a video introduction to In-Cell Western Assays – for those that like the movies!
Updated October 6, 2016.
IRDye BoneTag optical imaging agents are tetracycline derivatives that incorporate into mineralizing bone. Structural imaging of bone can be used to more precisely localize an area of disease. A second disease-specific targeting agent with a spectrally-distinct fluorescent label can be used to localize and track disease (such as a tumor) in the same animal. When the two images are overlaid, bone structure is displayed in one color and the other target appears in a different color.
IRDye 680RD BoneTag and IRDye 800CW BoneTag are part of the ready-to-use BrightSite™ optical agents family and make it easy to begin animal studies immediately. These bright fluorescent agents are labeled with IRDye fluorophores for NIR fluorescence optical imaging, and they target a variety of disease characteristics. Simply administer the agent, then image with any small animal imaging equipment with appropriate 680 nm or 800 nm filter sets. No engineered cells or animals are needed.
Figure 1. IRDye 680 BoneTag agent for imaging of bone structure and remodeling. Tetracycline-derived probe reveals skeletal structure, and signal is stable for weeks. Dorsal view of mouse imaged with IRDye 680 BoneTag. Image acquired with Pearl® Small Animal Imaging System.
We’ll be at AACR in Chicago, April 1 – 4, Booth 3800. Stop by and talk to us about how you can start your small animal in vivo imaging experiments today.
PSVue® 794 is a near-infrared fluorescent probe for detection of apoptotic and necrotic cells, bacteria, and other anionic membranes. The compound exhibits fluorescence excitation maximum at 794nm and emission maximum at 810 nm and through its zinc(II)-dipicolylamine (Zn-DPA) moiety, it has been found to bind strongly to negatively charged bacterial cell walls (e.g. S. aureus, E. coli) and necrotic regions present in various tumors (e.g. mammary, prostate, glioma) in vitro and in vivo. In particular, it has also been found to bind to the phosphatidylserine (PS) residues exposed on the cell surface of apoptotic cells, making it a more cost-effective alternative to fluorescently-labeled Annexin V in various cell death assays.
Figure 1. MPTP was used to induce cell death in mouse brains as a model for Parkinson’s Disease. C57BI/6 mice were treated with MPTP to selectively destroy dopaminergic neurons. Mice were then injected with PSVue dye or control dye and imaged on the Pearl® Imager 68 hrs post injection. A. control (i.e. non-targeting) dye; B. and C. PSVue dye; D. excised brains from the three animals.
Download a scientific poster presenting information on the use of PSvue 794 in studying Alzheimer’s Disease, Parkinson’s Diesase, and contact dermatitis in mouse models.
For more near-infrared fluorescent probes, learn about BrightSite™ Small Animal Imaging Agents and CellVue® Burgundy Fluorescent and CellVue NIR Fluorescent Cell Labeling Kits.
LI-COR interviewed Dr. Go van Dam, a surgeon specializing in oncology at the Groningen University Medical Center in the Netherlands.
A key focus of van Dam’s research is to explore new tools such as targeted fluorescent imaging probes that will help address the challenges facing oncology surgeons. He discusses his research using near-infrared fluorescent imaging during surgery to improve cancer patient outcomes. Watch an interview with Dr. van Dam.
Vasilis Ntziachristos, PhD, Technische Universität München, Germany and Gooitzen M. van Dam, MD, PhD, University Medical Center Groningen, Netherlands presented “Shining New Light on Clinical Fluorescence Imaging” at World Molecular Congress in San Diego, CA in September 2011.