[Cytometry] CD4 purification

Howard Shapiro hms at shapirolab.com
Tue Feb 9 16:59:54 EST 2010

Is it your intention to study mRNA in CD4+ T cells? If so, you will need to separate them from the CD4+ monocytes/macrophages also present in PBMC. If you did this by positively selecting with CD3, you would stimulate the CD4+ T cells, so getting rid of the monocytes with CD14 and/or other antibodies that they express and CD4+ T cells do not would be a better bet. 


-----Original Message-----
>From: Mous Kim <kim.mous at ua.ac.be>
>Sent: Feb 9, 2010 3:48 AM
>Cc: cytometry at lists.purdue.edu
>Subject: [Cytometry]  CD4 purification
>Dear all, 
>Could anyone comment on the following please?
>I intend to perform mRNA expression analyses through real-time qPCR on CD4 cells purified with magnetic beads starting from frozen PBMC. I'd prefer purifying through positive selection since cell populations tend to be purer compared to negatively selected cells. I do not intend to stimulate the purified CD4 cells prior to putting them into trizol. Has anyone of you observed that cells became stimulated / that proteins/mRNA levels increased due to the CD4 positive selection? 
>Thank you in advance for your comments/suggestions !!
>Best regards,
>Cytometry mailing list
>Cytometry at lists.purdue.edu

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