Cell ELISA, Permeabilization cell - (Jul/09/2008 )

Hi.
I am starting to work with Cell ELISA, these are my condition of the protocol.
In a plate of 96 well culture the cell that express a chemokine receptor, the cells were treated with the appropiate agonist, in order to see the percentage of internalization of this receptor,, I have 2 conditions: 1. the cells in resting conditions and 2. with the agonist. But I would like to make a total quantification of the receptor on membrane also into the cytoplasm by ELISA, doing a permeabiliztaion of the cell. I made it, but the OD in resting cell is higher than on permeabilized cell. Is a bit contradictory Nooo????? I do not know, if somebody have worked with a ELISA Cell can give some information about it.

Ricercatore2008

Hi,

It sounds as if you're not using fluorescence. But I think you're problem would be solved if you did.

SURFACE RECEPTORS
Fix cells with 1% formaldehyde in PBS.

NOTE: Use Fresh reagent grade formaldehyde. You can get from pierce in convenient 10 ml ampules at 16% concentration. It does not contain methanol which is IMPORTANT. By fixing your cells with 1% fresh formaldehyde you will fix them without permeabilizing them or damaging the membrane in anyway...

TOTAL RECEPTORS
Fix cells in 4% formaldehyde in PBS. Also, use facs buffer (labeling buffer) containing 100 micro-molar triton-x

A 4% formaldehyde solution DOES permeabilize the cells. triton-x makes it easier for the antibodies to slip through the membrane. Doing both of these things is like wearing suspenders with your belt but it definitely gets the job done.

INTERNALZED RECEPTORS ONLY
Acid strip the suface of the cells with acetic acid and THEN Fix cells in 4% formaldehyde in PBS. Also, use facs buffer (labeling buffer) containing 100 micro-molar triton-x

This removes the receptors from the surface and leaves ONLY the receptors inside the cell...

If you have access to a flow cytometer I highly recommed you use FACS analysis. Your quantification will be more specific and (in my opinion) more reliable.

It's really not difficult...

I hope this helps.

Tim