More Essential Success Tips for Performing In-Cell ELISAs Using Non-Adherent Cells

Round Bottom Plates for In-Cell Western Assays

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.

  1. Are you using the recommended round bottom 96-well plate (BD Biosciences, P/N 353077)?
    1. 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.
    2. 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).
  2. Are you shaking or rotating the plates at a moderate to high speed?
    1. If yes, gentler shaking/rotating is needed to prevent cells from detaching. Cells will detach. Set shaking or rotating speed to very low speed.
    2. 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!