[Histonet] Processing lavages into cell pellets...

[Anthony Reilly]

Hi Jennifer
 
I realise there are products like Histogel about of which I have no knowledge however I have been using agar for over 30 years because I can get it easily and gratis from our microbiology department.
 
My experience is to spin down the cells, remove the supernatent, then add fixative.  The fixation helps to protect the cells from the heat of the molten agar.  Heat agar until just melted to ensure minimum temperature.  After adding molten agar spin again in a tube to give you a button of agar with the cells located at the pointy end.  After the agar has set the agar button can be removed from the tube and stored in fixative for processing.  Although there may be a small number of cells in the agar,  the agar does not process well on a short cycle(ie 2 hours)  Process on at least a 6 hour cycle and overnight is OK.  When embedding, the cells if in sufficient quantity, are visible in the agar allowing easy orientation.
 
All the best.
 
regards
Tony
 
 
 
 

Tony Reilly
Chief Scientist, Anatomical Pathology
Pathology Queensland-PA Laboratory
_________________________________________________
Clinical and Statewide Services Division| QueenslandHealth
 
Level 1, Building 15,Princess Alexandra Hospital
Ipswich Road,WOOLLOONGABBA  Qld4102
Ph: 07 3176 2412
Mob: 0402 139411
Fax: 07 3176 2930
Email: tony_reilly <@t> health.qld.gov.au
Web:  www.health.qld.gov.au/qhcss/
 
 


>>> "Johnson, Jennifer(Hist)" <Jennifer.Johnson <@t> genzyme.com> 16/03/2010 4:38 am >>>
Hi,

I am looking for a little advice from anyone who has experience
processing lavage fluid into cell pellets that can be sectioned and
stained.  I need to work out a protocol using lavages from knockout mice
that may eventually be used to look at human patients.  In both models
of disease I expect to collect a lot of macrophages.  



After reading, "googling" and looking up articles and the Histonet, I
have come up with the following "procedure".  I would appreciate advice
or ideas from anyone who has performed any part(s) of this type of
procedure:



1.       Collect lavage in PBS (perform actual lavage on euthanized
mouse using PBS as the diluent).  Store on ice until I get back to the
lab.



Question here - because I will be collecting many samples, should I spin
them right away or keep them on ice and spin them all at once?  Should I
add a mucolytic agent (mucolexx or the like) and then store them on ice
until I get back to my lab?  I know that in mice, although it will be a
model that has lung disease, mucus will not be a problem.  However, in
the human model of the disease it may be an issue.  I need to see the
effect of the mucolytic agent on the affected cells before using it on
the patients' lavages.  

Does anyone out there use a specific mucolytic agent that they like?  I
plan on trying 2 - the Mucolexx (MSDS only lists formaldehyde) and
Richard Allan Mucolytic agent (MSDS lists formaldehyde, ethylene glycol,
methyl alcohol and polyethylene glycol).  



2.       Spin down cells at 4C for 15 minutes.  Remove supernatant.  Add
fix - either overnight or for a few hours.  



Question - if I use a mucolytic agent that contains fixative -
formaldehyde - do I have to rinse it out (resuspend the cells in PBS or
other buffer and spin again) or can I just remove the sup and add a gel
(step 4)?  Is the mucolytic agent enough of a fixative (the
Thermo/Richard Allan and Mucolexx mucolytic agents contain 0.3 or 3 %
formaldehyde)?



3.       Once the cells are pelleted, should I fix them again or just
resuspend them in a gel (something like Histogel or an agar(ose)?  Does
anyone out there use either of these gels to look at cells?  Any
preferences?  



4.       Once the cells are in the gel, fix the cell pellet.  (Is this
necessary?)



5.       Pray and process the pellet into paraffin (and maybe plastic
too).



Thanks in advance for any help you can give.  



Jenn



Jennifer Johnson

Genzyme Corp.

Department of Pathology

5 Mountain Road

Framingham, MA 01701-9322

Phn - 508-271-3610

Fax - 508-872-9080



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