Re: [Histonet] bone marrow mush

From:Sharon Cooperman

Hi Patsy,

I think the viability depends on how long the cells were in the tube 
at 4C.  I harvest bone marrow by scraping or flushing it out of a 
bone and then I manipulate it, etc. for a few hours and the cells 
maintain morphology and viability.  You can also do metabolic 
labeling on bone marrow (the cells are still viable) for hours after 
harvesting it this way.  But if it's been 24 hours or more I would 
think the cells would be dead and autolyzing.  You should probably 
tell them to make a smear or put them into fixative after harvest if 
they're going to be on ice for a long time.  Another way to determine 
what the cell population in the sample is would be to do a western 
with cell specific markers for the populations of interest or FACS. 
But again, if the sample has been sitting at 4C for a long time I 
would think the cells would be dead and the proteins degraded, so no 
good for FACS or western.  If the sample was frozen at -20C it would 
probably be fine for western, though.


>I have a question for you.  One of my investigators harvested bone
>marrow by just scrapping it out of the bone cavity, it is not in
>anything (media, anticoagulant, fixative, etc.)  they just stored it at
>4dc as a mush in a tube.  They want me now to prepare it so that they
>can see what cells they have.  They also want to know if there are any
>stem cells and what the viability is (by viability I mean they are
>wondering if they could use it to grow some of the cells in culture).  I
>really don't think I can tell them if they can grow the cells or not.
>Anyway I was thinking of trying to make a cell block from this material
>and process into paraffin or maybe even do frozen sections on the
>pellet.  How would you go about making a pellet out of that stuff?
>Would you put some buffer or media or fixative in the tube and spin it
>down and then put it in HISTOGEL or wrap in tissue paper and then
>Patsy Ruegg
>Histonet mailing list

Sharon Cooperman       	     
NIH, NICHD, CBMB                     301.435-8417
Building 18T, room 101               301.402-0078 fax
Bethesda, MD 20892

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