Using Non-adherent Cells for Fluorescent Immunoassays – Tips for Successful In-Cell Western™ Assays

In-Cell Western Assays - Fluorescent Immunoassays

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).

  1. How do you make non-adherent cells (suspension cells) attach to plates?

  2. 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.

  3. How do I know that I have a monolayer?
    Method #1
  4. – 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.

  5. 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.

Are You Studying Phosphorylation or Quantitative Cell Signaling Analysis? How About for IC50 Determinations?

In-Cell Western Assays - Fluorescent Immunoassays

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.
Use of labeling for In-Cell Western Assay normalization.
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).

Dose titration of Wnt3a treatment of mouse L-cells.  An In-Cell Western Assay Application.
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!

Quantitative Cell Signaling Analysis – What’s all the Buzz About?

In-Cell Western Assays for Quantitative Cell Signaling Analysis

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.

Optimizing Chemiluminescent Western Blots – The Best Offense is a Good Blocker

Okay, it’s football season, and I thought the analogy fit. 🙂 Seriously, the right Blocking Buffer is critical to getting that great chemiluminescent Western blot.

Incubating the membrane in blocking buffer after the transfer step will result in enhanced sensitivity of your blot. Blocking buffer contains proteins that stick to the membrane, promoting specific binding of the primary antibody and minimizing non-specific interactions. Various blocking buffers are available, and it’s important to try several blockers to find the optimal solution for each antigen and antibody pair. There is not a best blocker for all conditions – so you will need to do some testing.

One very, very, very important thing to keep in mind is that the blocker used with HRP-conjugated secondary antibodies in the secondary antibody incubation step of chemiluminescent Western blotting cannot contain sodium azide.

Why?, you ask.

Well, sodium azide binds irreversibly to the HRP enzyme, inhibiting the binding of the substrate and slowing the chemiluminescent reaction. This results in less light production that may affect the appearance of less intense bands or even the entire blot. See the figures below – the blot on the left was done with blocker that did not contain azide; the Western blot on the right used Western blocking buffer with azide.

Blocking buffer with and without azide

Nota bene: Odyssey® Blocking Buffer (PBS or TBS) (which does contain sodium azide) CAN be used to block the blot and to dilute the primary antibody but not to block or dilute in the secondary antibody incubation step when using HRP-conjugated secondary antibodies.

On Another Note: Milk is a common blocking buffer; however, milk-based blockers that contain endogenous biotin and glycoproteins may result in higher background on the membrane when detecting with streptavidin. Milk may also contain active phosphatases that can de-phosphorylate phosphoproteins on the membrane.

Maximizing Sensitivity on Chemiluminescent Western Blots Technical Note

Go Digital with the Odyssey Fc Chemiluminescent and Infrared Fluorescent Imaging System or the C-DiGit® Chemiluminescent Western Blot Scanner!