Greg,
Sorry to all those who aren't interested in this thread on the mailing
list, the following response is fairly long.
In response to your questions, I have several things to recommend. It
is not recommended to use purified antibodies for intracellular staining if
a direct conjugate can be found. This is mainly due to background
associated with the use of secondary antibodies and/or streptavidin (if you
are using a three step method.) Most (can't say for all) vendors do not
screen their secondary reagents for intracellular staining, and quite a few
antibodies either bind to intracellular epitopes, are bound by internal
receptors/factors or are generally non-specifically binding to the interior
of a cell. This does not mean that you can not distinguish true
cytokine-producing cells using a two- or three-step stain. Calman Prussin
has shown very nice data using biotinylated antibody staining for
intracellular cytokines. I often have to screen new clones as a purified to
determine whether or not to investigate it as a conjugate, and use a
biotinylated secondary antibody and either streptavidin-PE or APC as a
third step in order to distinguish cytokine staining above
secondary/tertiary reagent background.
So to answer some of your questions: 1.) That depends on what you
consider background. Using the staining of your secondary antibody alone
would be a good background control, with the theory being that increased
staining you get when adding the cytokine specific antibody would be
positive staining. I could also either pre-block the antibody with
recombinant cytokine which should preclude any staining with your antibody
leaving any staining you detect to be background. It is a difficult
question to answer.
2.) It might be best to go with a single source for the reagents, but
shouldn't really matter as long as the antibodies you have purchased have
been screened using the same fixation/permeabilization methods.
3.) There is a difference in the amounts of antibody one might use for
intracellular staining of cytokines versus the amount used to stain CD3 or
another CD marker. This has to do with both the higher propensity for
background staining when intracellularly staining cells and for the lower
number of epitopes being stained. I see amounts differing by 1/2 to 1/4 or
greater depending on the antibody (This is using a direct conjugate, using
a purified antibody usually requires more antibody for better results.)
4.) It is not unusual to have differing responses between individuals.
I find widely varying stimulation results even on cells which have been
activated using a 5 day in vitro differentiation culture, ie. one donor
having 5% IL-5 production versus another producing 0-0.5%.
As for surface blocking of Fc receptors, I have found little need to
block Fc receptors on in vitro activated PBMC cultures when I am later
fixing/permeablizing the cells and staining with PE-conjugated
anti-cytokine antibodies. It may be that a direct conjugated antibody will
help you out in more than one regard.
The only other suggestion I can give is that there are a wide variety
of suppliers of direct conjugated anti-cytokine antibodies, be sure to
contact them and see if the antibodies work in a staining protocol you are
familiar with or if they require special treatments. Not all antibodies
will stain fixed epitopes, so if you are using reagents which are sold for
ELISA, they may not be useful for flow cytometry. Hopefully this will make
things easier on you.
If you have anything else I might help with, let me know. Good luck in
your assay.
Sincerely,
-Jerry Wilson
Research Immunology - Cytokines
PharMingen
A Becton Dickinson Company.
Greg Neely <ggneely@ucalgary.ca> on 03/11/99 12:18:49 PM
To: cyto-inbox
cc: (bcc: Jerry Wilson/SDCA/BDX)
Subject: Intracellular/extracellular cytokine detection using GAM-PE
secondary AB
Hello, My name is Greg, I am a graduate student at the university of
Calgary, Canada.
I do intracellular/extracellular flow cytometry for cytokines in human
PBMC. I do not have fluorochrome-labeled cytokine-specific antibodies,
so I use a two-step process. I have a few questions about how I have
been doing things, and nobody around here can give me good answers, so I
thought some of you may be able to help.
1. First off, what is the best control to use for this two step
staining. I have tried both isotype-matched primary followed by labeled
secondary and just secondary with no primary. Which of these (or other)
is the best way to ascertain background fluorescence?
2. My cytokine antibodies are from R&D, my isotype-match control is from
PharMigen. Should I try to match suppliers for my isotype-match or is
this not a big deal. It means I have to spend a couple hundred more
dollars, which I don
Ęt want to do, but I want to discern specific from
non-specific staining so I can trust my results.
3. I use btw 1.25 and 2.5 ug primary and 1 ug secondary per 1.5 million
PBMC. Does this concentration sound within acceptable ranges? I have
tried different concentrations, this appears to be the best range. Does
anyone know if surface or intracellular staining usually requires
different Ab concentrations?
4. Like I said, I work with human PBMC. I am looking at surface and
intracellular cytokines, and I have found a lot of variation between
donors. Some will respond to stimulus, some will not. Sometimes donors
have surface expression in freshly isolated PBMC, sometimes they don
Ęt.
What degree of reproducibility is acceptable when staining PBMC? I can
see general trends, but I want to know how much leeway to allow these
trends.
5. I just tried to stain my cells for surface markers in PBS, 0.1%
sodium azide, but 1% human AB serum instead of fetal calf serum, hoping
this might better block non-specific interactions. Instead my cell
count was absurdly low, and there was tons of dead cells. Has anyone
seen this? Is this why one does not traditionally wash/stain cells in
human serum?
I realize I have brought up a few different issues, so feel free to just
respond to individual issues,
Thankyou in advance,
Greg
P.S. I enjoy eavesdropping on the flow discussions that take place in
this group, and I have found them very helpful on many occasions, so I
want to thank who ever is responsible for this group
Ęs existence.
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