On Sun, 23 Jul 2000, Len Brown wrote: > What is the consensus as to "wash" or "not-to-wash" human blood cells after > Annexin-V FITC staining. Most protocols do not wash. If you wash in what > buffer, Annexin Ca++ binding buffer, PBS etc.? This subject has been touched > on briefly in the past but not resolved. Hi Len, I think that you are right in that most kits that use Annexin do not generally suggest washing although some do dilute with the annexin binding buffer before running on the cytometer. It is a bit of a balance between ensuring that there isnt any 'non-specific' fluorescence due to the annexin-fluorochrome still being in the tube even if unbound and losing cells - possibly preferentially losing apoptotic cells, after washing. In general, with the samples I use, I am more concerned with cell loss so dont wash and hope that the amount of background will be the same with all samples. I would be interested to hear how others do it though. > Secondly, if staining for apoptotic cells with PI after ethanol fixation and > subsequent analysis for a sub-G1 peak, do investigators apply the same > strict criteria as to CVs of G0/G1 peaks as if they were solely performing > cell cycle analysis, i.e. <3% for a diploid human lymphocyte population? To see a sub-G1 peak there has to have been DNA damage (by whatever route) and loss so I would have thought it unreasonable to expect the same criteria to be applied to the assessment of the G1 peak - even within the populations that show 'normal' DNA content there may have been DNA loss to a small degree as well as conformational changes which will affect the CV of the peak. In general I tend to use the sub-G1 method as a more qualitative than quantitative method, certainly I wouldnt suggest that ALL the apoptotic cells would appear there, its good to give you an idea that something is happening but than I would use other methods to get better quantitation. Best wishes, Derek ************************************************************************ Derek Davies Voice: (44) 020 7269 3394 FACS Laboratory, FAX: (44) 020 7269 3100 Imperial Cancer Research Fund, e_mail: derek.davies@icrf.icnet.uk London, UK mobile: 07790 604112 Web Page: http://www.icnet.uk/axp/facs/davies/index.html In tenebris lux *************************************************************************
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