[Cytometry] Isolation of lymphocytes from sheep lymph nodes

Elizabeth Washington eawash at unimelb.edu.au
Wed Jul 21 20:23:25 EDT 2010


Hi Tracey,

We have have always found 60-70% non-viable cells when teasing out sheep LN.

Like you, when I first moved from mice to sheep I was worried if our
technique was to blame.  We did a lot of tests making mouse and sheep LN
suspensions in parallel - testing how gentle we were (chopping and teasing
vs pushing through a sieve) and whether different PBS and media were to
blame.  I even doubted the water we used to prepare media between my
previous mouse lab and the sheep lab!  The upshot was that treated the same
way and at the same time, mouse LN viability was always good (around 90 %
viable) compared with the sheep LN cells.

We have since tested sheep LN suspensions using annexin V and SYTO 17 and
confirmed that there is massive apoptosis occurring in the sheep LN, both
before and after birth.  We are preparing these papers at the moment, but
out techniques are described in Holder et al, EJI 2006 36: 2624-2631.

One thing we always do and which makes a big difference with clumping is
include 0.4% EDTA in our collection PBS/FACS wash.

You could gradient-purify the viable cells, though sheep cells don't always
behave like rodent and human cells on gradients and you would need to test
if subsets are lost in the process.  Alternatively, adding DNAse to the
medium can reduce the stickiness.

We often use 7AAD as a dead cell marker, but the dead & apoptotic cells
mostly appear in a discrete population with lower FSC and slightly higher
SSC so that they can be gated out fairly easily on these parameters alone.

Let me know how you go and if you need more detailed protocols.

Cheers,
Liz


-- 
Dr. Elizabeth A. Washington
Laboratory for Foetal & Neonatal Immunology
Department of Veterinary Sciences
The University of Melbourne
Parkville, VIC 3010
Australia
Phone:      61 3 8344 7347
Fax:        61 3 8344 7374
email:    eawash at unimelb.edu.au



On 21/7/10 12:47 AM, "Tracey Lee-Pullen" <tracey.lee-pullen at uwa.edu.au>
wrote:

> Hi All,
> 
>  
> 
> Does anyone here have any experience isolating lymphocytes from sheep
> lymph nodes?  We have a group of researchers isolating cells from foetal
> lymph nodes using a method of mechanical disaggregation based on a
> murine protocol.  I've done a lit search (and mailing list archive)  but
> so far all the protocol descriptions I can find are fairly brief and
> none of the studies have used a live/dead marker.  Using a viability
> marker reveals a very large amount of cell death - 60-70% and shows
> substantial non-specific staining of the non-viable cells.  The
> researchers preparing the samples report that when they isolate these
> lymph nodes and directly stain the tissue with trypan blue they already
> see a large number of dead cells even before they begin the process of
> dissociating and staining the cells.  We are now trying to sort these
> cells for them but are finding they are extremely sticky and clumping
> which we suspect is due at least in part to the large number of dead
> cells.  
> 
>  
> 
> Does anyone have any suggestions to help us reduce the number of dead
> cells, or maybe remove the contaminating non-viable cells?
> 
>  
> 
> Thanks in advance,
> 
>  
> 
> Tracey
> 
>  
> 
> Tracey Lee-Pullen
> 
> Graduate Research Assistant
> 
> Centre for Microscopy, Characterisation and Analysis (CMCA)
> 
> The University of Western Australia
> 
> Mail Bag 510
> 
> 35 Stirling Highway, Crawley, WA 6009
> 
>  
> 
> Ph: +61 8 9346 4410
> 
> Email: tracey.lee-pullen at uwa.edu.au
> 
> Web: http://www.cmca.uwa.edu.au
> 
>  
> 
>  
> 
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