Human whilte blood cells, CD3 + CD28 stimulation

[Jayaraman, Sundararajan]

Hi,

It is an immunology question. As I understand it, you are stimulating PBMC
with anti-CD3 and anti-CD28 antibody and then restimulating with PMA +
ionomycin. This is a standard method for cytokine production in human
PBMC. I assume that you are stimulating PBMC with immobilized anti-CD3. In
that case, you do not need soluble anti-CD28 antibody. It is only needed
when you have purified T cells and no accessory cells are present in the
culture. Since PBMC contain monocytes, you do not need anti-CD28 antibody
stimulation.

The second issue is that you are restimualting cells presumably after 4 to
5 days of culture in the presence of recombinant IL-2. If you are
stimulating with PMA and ionomycin in the presence of Brefeldin A for 4
hours (standard time), you should be able to stain intracellular
cytokines. It is well known that PMA can downregulate the surface
expression of CD4. However, you can stain the intracellualr CD4. For that,
permeabilize the cells and then stain for CD4 and intracellular cytokines.
  Alternatively, you can surface stain with anti-CD3 antibody,
permeabilize and then stain for two intracellular cytokines
simultaneously. You can do a 3-color analysis without much of a problem. 
We have done this successfully.

One important note is that you should use isotype controls when you fix
and permeabilize cells and use for intracellular cytokines. Unlike what
has been proposed, we find that isotype controls help to set the
background signal since fixation and permeabilization facilitate the
nonspecific binding of antibodies and also increase the autofluorescence.

Hope this helps.

S. Jayaraman, Ph.D.
Associate Professor of Surgery
University of Illinois at Chicago
909 South Wolcott Avenue-704E MSB-M/C 790
Chicago, IL 60612
Phone: 312-355-5133
Fax: 312-355-1497



On Wed, February 6, 2008 8:33 am, Simon Monard wrote:
> Do you have CD8 cells? Is there and complement there? Do you inactivate
> any
> serum you use? After how long do your cells disappear? My money would be
> either, complement lysis of CD3+ cells or T cell death following apoptosis
> induced by the anti CD3 ab, the CD28 not doing its job.
>
> Simon
>
> Quoting hanifi <ah200259ha at yahoo.com>:
>
>> Dear All,
>>
>> I am trying to detect cytokines with flow in T cells, I have white blood
>> cells and
>> stimulate them with CD3 and CD28, I also do restimulation(PMA, Iono) and
>> use
>> an inhibitor
>> but the issue is that the stimulated cells(comparing to non-stimulated)
>> have
>> no CD4+
>> cells. I do double staining for cytokines and CD4. I appreciate it if
>> someone
>> could tell
>> me what is going on, why do the cells die, what should I do,
>> Thanks
>> Arezoo
>>
>>
>>
>>
>>
>>
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>
> --
> Simon Monard
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