Re: Human platelets for FACS analysis

Hi Albert,
	I normally use E00 scale with FSC & SSC on Log; the rbc are 
then in the top right corner (depending on the voltages used)  with 
the platelets as a diagonal starting from the lower left corner of 
the rbc cloud going into the noise cloud in the lower left hand 
corner - how much of this appears depends upon how high your 
thresholds are set. As for ab labelling I normally use 1ug per 25ul 
of platelet prep, then dilute up to run on the analyzer.

Regards

Gary

At 10:01 AM -0700 9/16/05, Albert Tai wrote:
>Dear Flowers,
>
>I just start working with human platelets and have no prior 
>experience with it.  In my first experiment looking at rTK on 
>platelets, I was not sure if I used the correct FSC and SSC.  I set 
>both FSC and SSC in log scale and the gain was E02.  I saw a large, 
>square population at lower right corner (assembly platelets) and a 
>small population at upper right corner (rbc ?)  I also tried 
>lowering the gain to E01 as suggested in the archive but did not see 
>a diagonal center population.	Any insight on the parameter for 
>looking at human platelets would be appreciated.
>
>The rTK Ab we used was recommended at conc. 2ug/ml for 1e9 platelets 
>and it seems low to me but your expertise would be helpful in terms 
>of a general rule of thumb for primary Ab to platelet ratio.
>
>I would also appreciate any recommendation on reviews or 
>publications on the topic.
>
>Thanks very much in advance for your help.
>
>Best regards,
>
>Albert Tai
>Exelixis Inc.


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Dr. Gary Warnes
FACS Laboratory
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