Application: Detecting phospho-p53 in COS cells in response to Hydroxyurea
In this In-Cell Western assay application, the response of COS-7 cells to increasing doses of hydroxyurea was measured by a specific antibody (Anti-phospho-p53 from Cell Signaling Technology, P/N 9286) that detects phosphorylated-p53 (Ser16). Total ERK1 was used for normalization. The image represents a 96-well two-color In-Cell Western with the 700 and 800 nm channels detecting phosphorylated-p53 (Ser16) and total ERK1, respectively. Background wells were incubated with secondary antibody but no primary antibody. IRDye® 680RD secondary antibodies were used for detection in the 700nm channel and IRDye 800CW secondary antibodies were usd for detection in the 800nm channel.
The graph represents the average of four sets of quantitative data, demonstrating the percent induction of phosphorylated-p53 (Ser16). Plate-based assays such as this can be imaged on the Odyssey® CLx or Odyssey Sa Infrared Imaging System.
For more uses of In-Cell Westerns Assays, visit our website.
If you have been following my posts for the past two months or so, you know we have been looking a great deal at In-Cell Western Assays (also called In-Cell ELISAs or plate-based immunofluorescent assays). On April 3, I explained what an ICW Assay is. From there we looked at examples of how In-Cell Westerns are used (studying apoptosis, for IC50 Determinations), seeding plates for In-Cell Westerns), ICW kits LI-COR® offers, and using cells in suspension, plate selection for ICW Assays, and cell lines that have been tested for use with this powerful immunocytochemical assay.
So you are hopefully ready to give this technique a try. When you do, it is important to assess the overall quality and reliability of the assay during In-Cell Western (ICW) assay optimization. The Z’-factor statistic provides a way to evaluate whether or not assay conditions (reagents, protocols, instrumentation, kinetics, and other conditions not directly related to the test compounds) are optimized. Z’-factor, introduced by Zhang et al., is a dimensionless value that represents both the variability and the dynamic range between two sets of sample control data.
Z’-factor experiments are performed on one or more ICW assay plates containing replicate wells designated for background subtraction, negative control samples, and positive control samples. Typically, negative control wells are those in which the cells receive an the appropriate treatment so as to elicit the lowest desired percent response (usually untreated cells); positive control wells are those in which the cells receive an appropriate treatment so as to elicit the maximum desired percent response; background wells are treated the same as the negative control wells, except primary antibody incubation is excluded.
Here are some resources so that you can read more about the Z’-Factor, how to set up the experiments to assess it, and its importance in ensuring you have a high quality, reliable assay method:
Using the Z’-Factor Coefficient to Monitor Quality of Near-Infrared Fluorescent Cell-Based Assays
In-Cell Western Assay Quality Assessment Using Z’-Factor Data Sheet
Read more about Reproducibility and Precision in In-Cell Western Assays.
So I’ve talked about how to ensure that suspension cells attach to plates, when to know you have a monolayer, and why round bottom plates are the best when doing In-Cell Western Assays with suspension cells in the 23-May-12 blog post. On 29-May-12 post, the post discussed how to wash your microplates so that you don’t lose cells plus some troubleshooting tips. I hope you found both of those posts helpful.
Here is the last post in this series on using non-adherent cells for ICW assays. These are a few additional questions you may have about using suspension cells for this powerful immunofluorescent assay.
- What suspension cell lines have been tested for use in In-Cell Westerns?
Suspension cell lines tested include Jurkat, K-562, and THP-1.
- What pathways have been tested?
Pathways tested include ERK activation and apoptosis using cleaved caspase-3 as a marker (Figure 1). A sample protocol can be downloaded here.
Do you have other questions? Super! Please contact us and let us know how we can help you in your research. And stop by this blog again for more technical tips and troubleshooting hints on other applications.
Figure 1. Anisomycin-induced apoptosis in Jurkat cells. The image represents a 96-well two-color In-Cell Western assay with the 700 and 800 nm channels detecting TO-PRO®-3 DNA staining and cleaved caspase-3 (Asp175), respectively. The image was scanned using the Odyssey® Sa Infrared Imaging system with scan setting of 200 μm resolution, focus offset of 3.5, and intensity of 3.5 (700 channel) and 4 (800 channel). Background (B) wells were incubated with a secondary antibody but no primary antibody. The graph represents normalized quantitative data demonstrating the increase in caspase-3 cleavage in response to anisomycin treatment for 3 hours in Jurkat cells.
In-Cell Western Assays: FAQs when Using Suspension Cells
Complete Sample Protocol for PMA-Induced ERK Activation in Suspension Cell Lines
LI-COR BIO Website
I mentioned in my post on 23-May, that the next few entries would be on more hints and tips of how to use non-adherent cells for In-Cell Western Assays – so here goes!
During my washing steps, cells are coming off the plates.
- Are you using the recommended round bottom 96-well plate (BD Biosciences, P/N 353077)?
- If no, cells will more easily detach from the flat bottom plates than the round bottom
plates. The multi-channel pipettors will generate enough pressure when expelling liquid from the pipet to cause cell detachment when using flat bottom plates. Cells will detach even when pipetting down the sides of the wells.
- If yes, make sure you pipet down the sides of the wells and not directly onto the cells. If this doesn’t help, you may need to change your multi-channel pipettor because different brands of pipettors have different amount of pressure required to expel the liquid from the pipet. The recommended multi-channel pipettor is the 12-channel Finnpipette
(Thermo Electron Corp, P/N 4610050).
- Are you shaking or rotating the plates at a moderate to high speed?
- If yes, gentler shaking/rotating is needed to prevent cells from detaching. Cells will detach. Set shaking or rotating speed to very low speed.
- If no, are you dumping the solutions straight from the plates? Dumping causes cells to
detach. Either aspirate very slowly or manually pipet using the sides of the wells.
Why can’t I use the flat bottom 96-well plates?
- LI-COR® Biosciences recommends using the round bottom 96-well plates for the reasons mentioned above.
When I scan an empty round bottom 96-well plate, I get lots of background noise.
- The round bottom plate shows some background autofluorescence. The background fluorescence is relatively small compared to signal (about 200-fold difference depending on the intensity of the signal) and can be subtracted from the signal. It is necessary to include background wells containing cells and only the secondary antibodies in order to
completely subtract away the background noise originating from the plate as well as from the non-specific binding of the secondary antibodies.
Here is a technical note on more FAQs on using non-adherent cells for In-Cell Western Assays. Or you can just stay tuned to my next blog post!
To your Research Success!
You might be wondering if this powerful technique called In-Cell Western Assay can be used for your cell line because your cell line is non-adherent. Well, you are in luck! You CAN use suspension cells for ICW Assays – with some care and optimization.
Here are a few frequently asked questions. (see my next few blogs for more FAQs on using suspension cells for In-Cell Western Assays).
- How do you make non-adherent cells (suspension cells) attach to plates?
A simple trick is to replace your complete media containing 10% serum (usually fetal bovine serum) with the same media minus the serum. Then allow the cells to sediment, forming a monolayer of cells within 10 minutes. Caution: Although cells appear attached to the plates, they are relatively loosely attached and therefore, extreme caution is required during solution-changing steps.
- How do I know that I have a monolayer?
– Examine cells in the round bottom 96-well plates under a light microscope. The center of the wells should all have a small flat circular surface area where all the cells in that field are “in focus”. Moving the plane of focus, up or down, will cause cells to be “off focus”.
Method #2 – Hold the round bottom 96-well plate under a light source. The monolayer should look opaque rather than transparent. Cells will not attach on top of the cell monolayer, so the opaqueness is due only to the monolayer.
- I cannot get a monolayer of cells. I get lots of spaces between cells. Is seeding 200,000 cells/well enough?
Seeding 200,000 cells/well is more than enough to form a complete cell monolayer. It is necessary to allow the cells in serum-free media to sediment in the T75 flask (or other tissue culture plates) for approximately 30 minutes before counting cells using a hemacytometer. When cells in serum-free media are placed, for example, in a T7 tissue culture flask, a monolayer of cells will immediately begin to form on the bottom of the flask. This will dramatically decrease the number of cells in suspension that are available for plating.
Note: Once a complete monolayer has formed on the plate, the rest of the cells will remain in suspension. Count these cells in suspension and the cells attached to the T75 flask can be discarded later.
Here is a complete sample protocol for PMA-induced ERK Activation in Suspension Cell Lines.
Check on the website pages on Tips for Using Cells in Suspension Cells for In-Cell ELISA Assays.
What is an On-Cell Western? It’s a cell-based assay that enables quantitative monitoring of cell surface protein expression. The On-Cell Western assay offers the ability to:
- Detect and quantify target proteins localized to the cell surface
- Quantify ligand binding to cell surface receptors
- Monitor receptor internalization and recycling by detecting loss and re-appearance at the cell surface
- Perform and detect cell surface biotinylation assays
- Evaluate the effects of mutations, drugs, and other treatments on protein trafficking
- Analyze many samples quickly and quantitatively
- Avoid use of radioactivity
Figure 1. Cannabinoid receptor 1 (CB1) is internalized after exposure to a specific agonist (Win-2), but the effect is blocked by the antagonist SR1. A) Intensity levels were greatly reduced in wells treated with 1μM of the CB1-specific agonist, Win-2. Cells treated with Win-2 and the specific CB1 antagonist, SR1 displayed no reduction of signal with the treatment. B) Graph displaying results of three independent experiments done in quadruplicate.
Reprinted with permission from Miller, J.W. Tracking G protein-coupled receptor trafficking using Odyssey imaging. LI-COR Biosciences application note (2004).
Visit On-Cell Westerns for more information. We have a sample protocol of On-Cell Western Assay for Targeted Near-infrared-labeled optical imaging agent development. For other scientific publications in which On-Cell Westerns were used, visit our On-Cell Westerns Publications page.
Updated February 18, 2015.
The In-Cell Western™ Assay is an immunocytochemical assay that uses near-infrared fluorescence to detect and quantify proteins in fixed cells. Detecting proteins in their cellular context increases quantification precision. Proteins in fixed, cultured cells are detected directly in microplates, which yields higher throughput compared to Western blotting and eliminates typical Western blotting steps such as cell lysate preparation, electrophoresis, and membrane transfer. Using the In-Cell Western Assay kits, the cost per well for secondary screening is reduced to a fraction of the cost of typical screening methods. Watch an introductory webinar to In-Cell Western Assays.
The CellTag™ 700 Stain ICW Kits provide antibodies, blocking buffer, and CellTag 700 Stain to normalize well-to-well variations in cell number for forty 96-well plates or ten 384-well plates. Using protein stains reduces the cost per assay compared to performing the assay using two secondary antibodies. Any potential interference caused by using two antibodies is also eliminated.
Other In-Cell Western Assay Protocols are available on our Technical Resources Library. Visit our In-Cell Western Application Pages for more examples of how researchers have used these immunocytochemical assays.
Updated February 18, 2015.
LI-COR® has just rolled out a new way that the recently-released Quick Western Kit – IRDye® 680RD (see Would you like to save at least 90 minutes the next time you do a Western blot?) can be used in your research.
Not only can the Quick Western Kit reduce Western blotting time by 90 min, the kit ALSO serves as a detection solution for post-immunoprecipitation samples by Western blot because it does not bind to denatured mouse monoclonal or rabbit monoclonal antibodies. The key benefit is the ability to use the same antibody for immunoprecipitation and post-immunoprecipitation detection by Western blot. Seriously, how cool is that!!??!!
Figure 1. A431 cell lysates were immunoprecipitated overnight with a monoclonal antibody against p53. The resulting immunoprecipitates were separated by SDS-PAGE. Lane 1: Negative IP control; Lane 2: Test sample ; Lane 3: A431 cell lysate positive control. Western blotting was performed using the same p53 monoclonal antibody and incubated with IRDye 680RD Immunoprecipitation Detection Reagent.
Protocol: Detection of post-immunoprecipitation proteins by Western blot using the Quick Western Kit – IRDye 680RD
For more information, visit our website. Here’s the kit pack insert. To order this product (online ordering available in select countries), go to our ecommerce site.
In a previous post, I talked about how In-Cell Western™ assays could be used when studying apoptosis. So, you may be asking yourself, for what other applications can quantitative cell signaling analysis be used? GREAT QUESTION!!
Well, In-Cell ELISAs (as these immunofluorescent assays are also called) have been used successfully in studying protein phosphorylation. Whether you are looking at the effects of drug compounds on signaling pathways, or the timing/kinetics of signal transduction, or trying to determine the IC50 of compounds, In-Cell Western assays are a valuable tool.
Here are two examples of data from IC50 and EC50 determination experiments.
Figure 1. Use of cell labeling for In-Cell Western normalization. A) HeLa cells were treated with increasing amounts of rapamycin in a 384-well format. Fixed cells were stained with phospho-rpS6 antibody and NHS-ester reactive dye (for cell number). Dose dependent inhibition of phospho-rpS6-staining yielded an IC50 of 224 pM (n=4). B) Raw microplate image. For details, see Hoffman, GR et al. Assay Drug Dev Tech 8(2):186-99 (2010).
Figure 2. Dose titration of Wnt3a treatment of mouse L-cells. Half-maximal activation (EC50) of cellular beta-catenin levels occurs at 33 ng/ml ligand. Hannoush, RN. PLoS One. 3(10):e3498 (2008). Creative Commons license 2.5.
To help you get started in designing your experiment, here is a complete sample protocol for measuring IC50 of the inhibitor PD168393 in A431 cells responding to epidermal growth factor (EGF).
Check here for future blog posts on other applications of quantitative cell signaling analysis!
One of the first steps in an In-Cell Western Assay experiment is to seed cells into the wells of a tissue culture microplate. Cell density is more important for some cell lines than others. In particular, cells that depend more on extracellular activity for proliferation (such as epithelial cells) are affected to a greater extent by initial growth conditions. There are three factors to consider when seeding cells:
- Plates: For most adherent cells that stick to wells tightly (e.g. A431, HeLa, HEK293, CHO), we recommend regular tissue culture microplates with low auto-fluorescence, such as Nunc P/N 167008. For adherent cells that could detach from wells during In-Cell Western assay wash steps (e.g. NIH/3T3), we recommend Poly-D-lysine coated 96-well microplates.
- Cell seeding density: Typically, 15,000 to 40,000 cells are seeded per well. Two to three days are usually required for cells to reach the appropriate confluency, depending on growth rate. Seeding with low cell numbers is recommended if you plan to culture for several days before use. Plates seeded with higher cell numbers will be ready to use earlier.
- Confluence: To obtain maximal fluorescent signals, complete or near complete confluency is recommended for cells that stick to wells tightly. For cells that adhere loosely to wells, such as NIH3T3, 70% confluency should be used. Please note that cell type and experimental conditions may affect the acceptable level of growth confluency.
The example below illustrates the importance of cell seeding density for A431 cells. As shown in the corresponding graph, cell growth is greatly inhibited when there are too few neighboring cells.