Summary Document

This document is a summary of message from the Purdue University Cytometry Laboratories Cytometry Discussion list.

http://www.cyto.purdue.edu/hmarchiv/index.htm

Dear flowers,

Here is a synopsis of the responses I received to my original query about the expression of Fc receptors on Embryonic Stem Cells and the use of Fc blocking

reagents. Thanks very much to all who responded.

Patricia Lovelace Manager, Flow

Cytometry Geron Corporation 230 Constitution Drive Menlo

Park, CA 94025

1.   I have not had any problems (background staining) with mouse MEFs so far.  For routine Fc blocking when using mouse tissue samples, I incubate the cells with 2.4G2 (anti-Fc) for 20-30 minutes on ice.  Usually, I do not even wash the cells after blocking (a quick spin for removing 2.4G2 supernatant).  It works really well for me.  Ocassionally, in my liver MNC preparations, I do get background B cell staining inspite of 2.4G2 block.       I have not got around this problem yet (other than using a negative B220 gate). 

I have not used the Fc block after the first blocking step.  I block the cells first with the anti-Fc antibody, surface stain and directly fix and permeabilize my cells for intracellular staining.  Hope this helps.

Sriram

Venkataraman Sriram, PhD

Postdoctoral Fellow

The Walther Cancer Institute

Indiana University School of Medicine

Indianapolis, IN 46202

2.   In our hands we use a routine staining buffer (PBS) that includes Fc block in all staining and washing steps. I use 2.5% total protein in the block, 1% BSA,1% FBS and .5% nmIg. It can be pricey if you use at the recommended concentrations we are used to so we grow it up with a hybridoma Ig spitting cell line from ATCC ; MOPC-31C for staining mouse lymphocytes at 500ug/ml.  The Bible for my generation was and is Harlow and Lane, Antibodies-A Laboratory Manual, Cold Spring Harbor Press for all this kind of stuff..

Gene Pizzo

Manager, Flow Cytometry Facility
UCONN Health
Farmington, CT. 06032
http://flowcytometry.uchc.edu
860 679 7567

3.   What about doing a blocking step with 10 mg/ml IgG before staining and then not worrying about it?  We’ve done that for years, buying the cheapest IgG we can find—goat, horse, whatever.  And we block every cell type, because background staining can be due, not to

Fc receptor binding (you can eliminate that by using F(ab’2) fragments or biotinylated primary Abs + streptavidin-fluorochrome conjugates) but non-specific protein-protein interactions.  As far as I know, only certain hematopoietic cells normally express Fc receptors, but every cell has the potential to have protein stick to it.  Sort of like running an ELISA or western without a blocking step—you get high backgrounds there too. Sometimes we have to go to 20 mg/ml to reduce non-specific binding, but not often.

Beverly Barton

Beverly E. Barton, Ph.D.

Assistant Professor

Department of Surgery/Division of Urology

UMDNJ-NJMS  MSB G519

185 S. Orange Avenue

Newark, New Jersey 07103

Telephone 973-972-0662

E-mail bartonbe@umdnj.edu

Telefacsimile 973-972-3892