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.