[Cytometry] FACSCalibur prime problem

Haviland, David L David.L.Haviland at uth.tmc.edu
Mon Feb 1 17:58:23 EST 2010

If you aren't getting any backflushing through the chamber (yes, you have to take the covers off to see this) you might need to look a the waste line that the pump in the back works on.   You can do this without taking the covers off but I also set up a protocol and ask the machine to "acquire" (FSC/SSC) while you Prime it.  That way you can monitor cleaning and priming in real time.  

The big thing I'd like to know is if 1) when you hit prime does the flow cell purge, 2) when not priming, when you put the swing arm to the side, is fluid aspirated from a tube, and 3) if no tube is in place and you put the swing arm in the center position, sheath should dribble out of the SIP but be aspirated (a hanging drop works well for this) the minute the arm is moved to either side.    Depending on what you see, that black tubing and associated tubing that goes that pump you hear when you move the swing arm may need to be flushed out.  Number 2 and 3 are what should happen... if it doesn't, I'm flushing the tubing - if I can remove it... if not I carefully use a 5ml syringe with water and tenderly get an 18guage bevel of a needle into the tubing to flush it out. 

This also assumes your waste connecter (orange) that connects the electronic side to the wet side of the Calibur is still intact.   Bleach is hell on that connecter and I've had three connecters fail in 12 years and they tend to fail in the closed position.   If that connector is not working, you won't be able to move a single mircoliter through the instrument. 

Hope this helps... 


David L. Haviland, Ph.D.
Assistant Professor
University of Texas Health Science Center - Houston
Center for Stem Cell Research - Flow Cytometry Laboratory
The Brown Foundation Institute of Molecular Medicine for the Prevention of Human Diseases
1825 Pressler St., R637I
Houston, TX  77030
713-500-2413 Office
713-500-2424  Fax
Flow Center Website in progress

-----Original Message-----
From: cytometry-bounces at lists.purdue.edu [mailto:cytometry-bounces at lists.purdue.edu] On Behalf Of Paul Kron
Sent: Monday, February 01, 2010 11:19
To: Cytometry Mailing List
Subject: [Cytometry] FACSCalibur prime problem

Hi everyone.

I've been having trouble recently with high CVs on our FACSCalibur.  
Repeated cleaning and priming usually resolves the problem. I usually  
do a special priming step in which I vent the tank pressure as the  
cell starts to refill, then follow it with a "normal" prime (I can't  
recall what the name is for this type of a priming). Because this  
involves watching the cell drain and refill, I've noted the  
following: the cell is draining more slowly than it used to, and  
sometimes it does not drain at all (i.e. it is not actually priming,  
even though the prime light is on).

What could be causing this problem? I'd like to prepare myself for  
the prospect of a repair bill.

Paul Kron
Integrative Biology
University of Guelph

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