single cell sorting
mcabanas at gemabiotech.com
Mon Sep 17 15:52:28 EDT 2007
I use Moflo for single cell sorting and have no problem. You need to set the
Cytometer on Single Mode, and 0.5 Drop Criteria to ensures you are sorting
0-1 cell per well. Also make a FSC/ Pulse Width Plot and make a gate to
exclude the doublets/triplets. In the FSC/SSC Plot, make a gate inside the
population region, to pick from cells very similar. (homogeneous
To validate the single cell sorting, first I sorted Fluorescent Beads that
were the same size than the cells I wanted to sort.
I use 96 well plates with the cover on the plate. I use the covers that have
a circle corresponding to each well. So, after sorting one bead per well on
the plate (with the cover on), I take the cover and look at each "circle" in
an epifluorescent microscope. If it´s hard for you to make focus, you can
put the cover upside down on the microscope (the covers have some stoppers
and the plate surface will not touch the platin of the microscope). Then you
should go well by well and check that all of them have 0-1 bead. Right after
you have checked one bead per well you should sort you cells.
You can also test this by sorting the same Cells expressing GFP and checking
them by microscopy.
From: Liza Bronner [mailto:lbronne at emory.edu]
Sent: Friday, September 14, 2007 3:44 PM
Subject: single cell sorting
I am working on single cell sorting on a FACSAria. I was wondering if
anyone has any advice or tips or a protocol for single cell sorting.
How do I get just one cell per well? How do I count the cells that get
sorted into a 96 well plate to ensure that I get just one cell per well?
My clients are interested in single cell plate sorting for use with
PCR. Is a 96 well plate my best option as I do have access to smaller
I have sorted calibrite beads into a 96 well plate. I can tell the Aria
to sort 5, 10 , 20 events per well, but that does not mean that I get
this many beads per well. I have had difficulty counting how many beads
I get per well.
Thank you in advance for your help!
Research Specialist- Flow Cytometry Core
Emory Vaccine Center
954 Gatewood Road
Atlanta, GA 30329
lbronne at emory.edu office: 404.727.8619
Online Scheduler: evc-flowsched.rmy.emory.edu
-------------- next part --------------
HTML attachment scrubbed and removed
More information about the Cytometry