RE>>Intracellular % Formula I usually calculate % positivity using this formula: fluorochrome conjugated antibody blocked by un-conjugated isotype control - the staining blocked by un-conjugated antibody. Xifeng Yang -------------------------------------- Date: 2/27/97 7:36 PM To: cyto-inbox From: DODONNEL@svherc.ucd.ie Michelle Fiordalisi wrote "I calculate % positivity using BD's formula: (Activ. sample - activ. isotype control) - ( Unstim. sample - Unstim. isotype control)." I would be interested in other intracellular flow-ers opinion of this formula: do you use it widely? I have a problem with subtracting the unstimulated sample values. I presume tnat this is intended to allow for non-specific intracellular staining that can occur after permeabilisation. However, in some ex vivo situations where effector lymphocytes are already activated a small percentage of even unstimulated cells may be clearly positive relative to their isotype control. I doubt that it would occur in peripheral blood lymphocytes but I have certainly seen it for IFN-g in activated BAL cells in pulmonary disease. It occurred in situations where the clinical disease was highly active. By subtracting this percentage of cells from that in the stimulated sample one might be underestimating the total % of cytokine-expressing cells. What do the erudite people think? Dearbhaile O' Donnell, University College Dublin ------------------ RFC822 Header Follows ------------------ >From flowcyt.cyto.purdue.edu!owner-cyto-sendout Thu Feb 27 18:50:40 1997 remote from donews Received: from flowcyt.cyto.purdue.edu by donews.cts.com with smtp (Smail3.1.29.1 #5) id m0w0IP7-00005GC; Thu, 27 Feb 97 18:50 PST Received: by flowcyt.cyto.purdue.edu (940816.SGI.8.6.9/930416.SGI.AUTO) for cyto-sendout id RAA27536; Thu, 27 Feb 1997 17:30:01 -0500 Received: from hermes.ucd.ie by flowcyt.cyto.purdue.edu via ESMTP (940816.SGI.8.6.9/930416.SGI.AUTO) for <cytometry@flowcyt.cyto.purdue.edu> id LAA21339; Thu, 27 Feb 1997 11:19:12 -0500 From: DODONNEL@svherc.ucd.ie X-PMrqc: 1 Received: from svherc.ucd.ie by hermes.ucd.ie (PMDF V5.0-6 #16181) id <0E696Z72P000D5@hermes.ucd.ie>; Thu, 27 Feb 1997 08:45 +0000 (GMT) Received: from SVHERC/MAILQUEUE by svherc.ucd.ie (Mercury 1.11); Thu, 27 Feb 1997 08:45:13 +0000 (GMT) Received: from MAILQUEUE by SVHERC (Mercury 1.11); Thu, 27 Feb 1997 08:43:39 +0000 (GMT) Date: Mon, 27 Aug 1956 20:59:22 +0000 (GMT) Subject: Re: Intracellular % Formula To: cyto-inbox Message-id: <1F6F9887ECF@svherc.ucd.ie> Organization: University College Dublin X-Mailer: Pegasus Mail/Mac v2.02 Content-transfer-encoding: 7BIT Priority: normal
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