 |
 |
 |
 |
It sounds as though you have done the correct things:
1.) Titrate your blocking reagent (serum). Typically 5-10% serum in saponin
works. Block for 1h PRIOR to staining.
2.) Titrate your anti-cytokine mAbs. Use monoclonals, typically at 1-5
mcg/ml.
A few other suggestions that may help you:
-I assume you are working with PBMC from your note.
1.) Gate on lymphocytes by scatter. It should be obvious, as the
scatterplot is similar to unfixed cells. Monos have greater
autofluorescence and nonspecific binding.
2.) Try looking for IL-2 or IFN-gamma, as these are the easiest to start
with. I have no clue what cell type or cytokines you are interested in, but
lymphocytes are easiest. IL-2 and gamma IFN are very abundant in stimulated
PBMC.
3.) Use PMA/ionomycin as a stimulus. This combination works, once you have
this working, then try other more subtle stuff.
4.) Avoid cytokine expressing cell lines; in my experience autofluorescence
is usually greater and the positives are not so bright.
5.) Choice of mAbs-Critical!!! Ulf Andersson has published a list of mAbs
that work for microscopy (below).
At the risk of entering "commercialization of the flowNet purgatory" I
should add that Pharmingen has recently (last week) announced that they are
making a large number of their human and mouse monoclonal anti-cytokine
antibodies available in direct conjugate (FITC and PE) form. I have had the
chance to use some of their human reagents (anti-IL-4 and IL-5) with
excellent results. I have also conjugated some of my own mAbs (IL-2 and
IFN), also with great results. The decreased signal due to lack of a
sandwich step is more than offset by their low noise; hard to believe but
true. At present I have had more limited experience with their anti-IL-2
and IFN-gamma. PE seems to have greater S/N than FITC.
6.) James Jaccoberger recently wrote a chapter on intracellular staining in
"Methods in Cell Biology" vol. 41 page 351.
7.) We should be having a paper out in the May 1 J.Immunol. using cytokine
flow cytometry, revisiting the question of Th1 and Th2 cells. Check it out.
References: -Sander et al. Immunol. Rev. 119, p. 65 (1991)
-Andersson, J. et al., J. Immunology, 83, 16-24 (1994)
-Andersson, J. et al., Immunolabeling of cytokine producing cells in
tissues and in suspension in "Cytokine producing cells" eds. D.
Fradelizie and D. Emelie. INSERM, Paris. 1994.
Good luck,
Calman Prussin
Allergic Diseases Section
National Institute of Allergy and Infectious Diseases
National Institutes of Health
Bethesda, Maryland
Calman_Prussin@nih.gov
---------------------- Replied Message Body ----------------------
Date: 4-19-1995 2:18pm
From: {"Clive Gray" <CLIVEG@niv.ac.za>}:unix:niaid
To: calman prussin:10:niaid
Subj: blocking Fc receptors?
Also-to: cytometry mailing list <cytometry@flowcyt.cyto.purdue.edu>
--------------------------------------------------------------------
I hope someone can help!
We are trying a saponin permeabilization technique - to measure
intracytoplasmic cytokines and other proteins (p24 etc). We are
initially fixing with 4% formaldehyde and then coming in with saponin
and then obviously labelling with our Ab. However, when we label
fixed cells only there are very high backgrounds, even though our
isotypic controls are negative. I cannot believe that vimentin
(our positive control) resides on the surface of cells (PBMC). Does
this suggest that the monoclonal Ab is binding to Fc receptors? This
is also true for other monoclonals we are using - especially when
staining in vitro activated cells. We have scratched our heads and
tried blocking with goat, rabbit or horse serum before labelling as
well as tritating out our primary Ab. Does anyone have any experience
or ideas? Are we barking up the wrong tree? I would appreciate any
comments or ideas.
Thanks
Clive Gray PhD
University of the Witwatersrand
Johannesburg, South Africa
e-mail: cliveg@niv.ac.za
|
|
|
|