RE: staining and washing in Eppendorf tubes

Mara,
 
Since the ImageStream uses Eppendorf tubes as the standard sample
container,
we have quite a bit of experience with the format. I forwarded your
query to our
bio group and this is what Tad had to say:
 
If using a minifuge, we spin cells at 300xg for 5-10 minutes (same as in
the benchtop larger centrifuges) for all sizes of tubes.  Check the
minifuges instruction manual or website to determine what that
translates to in terms of rpm, as this number depends on the rotor
radius.  Avoid spinning really hard for short times as this potentially
damages the cells.
 
In terms of staining in eppendorfs, it is important that the wash steps
dilute the staining reaction 10 fold, so if you are staining in 100 uL
volume, you will want the add about 1 ml of wash buffer prior to the
spin (in this case use 1.5 ml tubes).  We typically stain cells at
concentrations between 1 and 3 x10^7 cells per ml (ie 1-3x10^6 cells per
100 ul).
 
Tad
 

Tad George

Principal Scientist

Amnis Corporation

2505 3rd Ave, Suite 210

Seattle, WA 98121

206-576-6859

t.george@amnis.com

 

Best,

David

 
David Basiji, Ph.D.
CEO, Amnis Corporation
2505 Third Ave., Suite 210
Seattle, WA 98121
(206) 374-7165 direct
(206) 919-3342 mobile
(206) 576-6895 fax
www.amnis.com
 


________________________________

	From: Mara.Rocchi@moredun.ac.uk
[mailto:Mara.Rocchi@moredun.ac.uk] 
	Sent: Tuesday, May 16, 2006 2:05 AM
	To: Cytometry Mailing List
	Subject: staining and washing in Eppendorf tubes
	
	

	Dear all, 

	Does anybody have suggestion on how to stain cells in eppendorf
tubes?

	I have been asked by one of our PI for a protocol that uses a
minifuge instead of a benchtop centrifuge.

	Which size of tube (0.2 ml, 0.5 ml, 1.5 ml, 2ml?) do you use and
at what speed and for how long do you spin?
	The staining is an intracellular single colour indirect staining
(2 steps).  

	 

	Thanks in advance for your help

	 

	Mara 

	 

	***********************

	Mara Rocchi 

	FACS manager

	Moredun Research Institute

	Pentland Science Park

	Bush Loan, Penicuik

	EH26 0PZ

	Scotland, UK

	Tel: 0131-4455111

	Fax: 0131-4456111

	e-mail:mara.rocchi@moredun.ac.uk

	 

	 



	The information contained in this e-mail message and any files
transmitted

	with it is confidential and may be legally privileged. It is
intended only

	for the addressee and others authorised to receive it. If you
are not the

	intended recipient or the person responsible for delivering the
message to

	the intended recipient you are advised that you have received
the e-mail in

	error and that any disclosure, copying, distribution or action
taken in

	reliance on the contents of the e-mail and its attachments is
strictly

	prohibited and may be unlawful. Please advise the sender
immediately by

	replying to this e-mail and delete it and all copies from your
system.



###########################################

This email and any attachment may contain information which is private 
and confidential and is intended for the addressee only. 
If you have received this email in error, please destroy it 
and notify the sender by return email.