single cell sorting

Maria Laura mcabanas at gemabiotech.com
Mon Sep 17 15:52:28 EDT 2007


Hi,

 

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
population). 

 

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.

 

Laura

 

 

 

-----Original Message-----
From: Liza Bronner [mailto:lbronne at emory.edu] 
Sent: Friday, September 14, 2007 3:44 PM
To: cyto-inbox
Subject: single cell sorting

 

Hello Flowers,

 

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 

Taraski plates?

 

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!

 

Best Regards,

Liza Bronner

 

-- 

Liza Bronner

Research Specialist- Flow Cytometry Core

Emory Vaccine Center

954 Gatewood Road

Atlanta, GA 30329

 

lbronne at emory.edu office: 404.727.8619

cell: 706.254.4654

 

Online Scheduler: evc-flowsched.rmy.emory.edu

 

 

 

 

 

 

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