intracellular cytokine detection

Aki Hoji akhst7+ at PITT.EDU
Wed Mar 11 00:05:35 EST 1998


>On Mon, 9 Mar 1998, frederic clement wrote:
>
>> Dear scientists,
>> 
>> Can anyone help me with commercial available permeabilisation kits or
>> other commercial stuff for detection of intracellular cytokines by
>> flow-cytometry ? 
>> 
>> Thanks a lot.
>> 
>> PS : Please mail me your advice.
>> 
>> 
>Several companies including Pharmingen (San Deigo) and Coulter sell kits
>to permeabilize cells.  However, inour laboratory we have found that the
>following procedure works quite well.
>
>1)  Surface stain as normal
>2) fix with 4%formaldehyde
>3) Wash with PBS
>4) add PBS containing 0.1% saponin and stain intracellularly for 30 min.
>5) Wash out intracellular stain with PBS-azide containing 0.1% saponin.
>6) fix with 1% formaldehyde
>
>This is the basic procedure.  The literature has published several methods
>based on this type of procedure
>
>Good luck
>
>Dr. Tim Wyant
>
>
Also,  an Appendix in a Pharnimgen catalogue describes a intracellular 
staining procedures.  Many of the commercially available kits nowadays 
come with Brefeldin A and/or Monesin which may or may not be effetive.  

regards, 

 


Aki Hoji (akhst7+ at pitt.edu)
Dept. Infectious Diseases and Microbiology
University of Pittsburgh




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