[Histonet] Processing lavages into cell pellets...

[Johnson, Jennifer(Hist)]

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