Re: staining and washing in Eppendorf tubes

Hi Mara-
Staining in microfuge/eppendorf tubes can really speed up your	
processing.
We routinely stain 100,000 to 1,000,000 cells in <100ul ;  and spins  
are about 14-18sec at 10,000g (depending on cell type, fixed or  
permed, etc).
Compared to 5-8minutes in the 5ml "FACS tubes"- it's a nice change.

(I will say- once you have more than 10-15 tubes to process  
simultaneously; time to look at staining in a 96 well plate- unless  
you have stainless steel thumbs!)

IMHO- it's best to work out the speed/timing for your specific rotor/ 
microfuge combo, and your particular cells.  (You didn't mention what  
type cells..)
For instance- when processing HUVEC, my spins are a shorter than when  
I stain PBMC's as the HUVEC's pellet faster.

Aliquot your cells into a couple of tubes, then compare  
recovery/"integrity" (are the cells easily resuspended- or now	
crammed into a tight snot ball?)
Ok, not the most scientific description- but a visual you will get  
right away if it happens...    I've used 3 different microfuge's and  
found they each had a "sweet spot" for best wash/ recovery. {An extra  
few seconds on one made a nice "PBMC puree"- even though I thought I  
had the same g force...}

I like the 2ml snap caps (I think I got from ISC?) instead of the  
"traditional" 1.7ml because: 1) the extra volume for washing and  2)  
the cell pellet  resuspends quite nicely.

If you prefer the longer/slower spins of a benchtop (600g, 5min) you  
could still do that in a microfuge tube. We've even set up mini-l- 
ficolls with 200ul blood!
good luck-
bunny



Bunny Cotleur, M.S.
Sr Research Technologist, Dept of Neurosciences
Scientific Consultant, Flow Cytometry Core
Cleveland Clinic Foundation
Lerner Research Institute, NC30
9500 Euclid Avenue
Cleveland, OH 44195
Lab: (216)444-1164
*********************************************
Caminante, no hay camino.      Se hace camino al andar.





On May 16, 2006, at 5:04 AM, Mara.Rocchi@moredun.ac.uk wrote:

> 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
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