Cells in Suspension for Quantitative Cell Signaling Analysis using In-Cell Western™ Assays

In-Cell Western Assays - Fluorescent Immunoassays

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.

  1. What suspension cell lines have been tested for use in In-Cell Westerns?
    Suspension cell lines tested include Jurkat, K-562, and THP-1.
  2. 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.

Anisomycin induced Apoptosis in Jurkat Cells

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.

Additional resources:
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