[Cytometry] Sorting Lamprey Cells

Sommer Durham sdurha4 at emory.edu
Thu Jul 16 16:55:11 EDT 2009


Hello Everyone,
  I was wondering if anyone has experience sorting lamprey cells. I am  
running the samples on an Aria II, and I usually utilize the 85 micron  
nozzle with 30psi pressure. Our problem is that our post-sort test  
samples show an unwanted population in the lower left corner of the  
scatter gate.  I tested using the 100 micron nozzle with 23 and even  
17 psi pressure to no avail. In addition, we tried using 1/3 PBS as  
sheath buffer and ended up with similar results.  I dilute samples to  
achieve a threshold rate of about 500 to 2000 events/sec at a flow  
rate between 1.2 and 1.8.  The cells are fairly small, but we tested  
with the 100 micron nozzle along with decreased pressure to try and  
decrease cell damage. We tried using 7-AAD or PI to ensure that these  
were dead cells showing up in our post-sort test samples.  The %PI+  
cells increased significantly, and the population located in the lower  
left corner of scatter are PI+. Sort samples are suspended in Ca+/Mg++  
free PBS containing 1%-2% FBS.  If you have recommendations on how to  
improve these results, let me know. Any input and advice on sorting  
lamprey cells will be highly appreciated, Thanks!


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Sommer Durham
Senior Technologist
Emory University School of Medicine
Flow Cytometry Core Facility
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Room 441
Atlanta, Ga 30322
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