[Histonet] FFPE cell pellet prep

[JR R]

Here's how I do it.

I hate the lens paper method.

I transfer suspended cells from a T75 flask to a 50 ml Falcon tube and centrifuge at 400 X g for 5 minutes.  Aspirate most of the media so that cells plus media are about 1 ml.  Transfer cells plus media to a 1.7 ml microcentrifuge tube.  Pellet cells at 400 x G for 5 minutes.  Rinse with PBS if needed.  Suspend cells in NBF and fix over night.  

Next day, pellet cells and resuspend them in a small amount (100-200 microliters) of Histogel or Agar.  When the gel cools cut the microcentrifuge tube open with a scalpel or razor blade.  Now you have a nice cell pellet shaped like the bottom of your centrifuge tube, and you can treat it just like any piece of tissue. Pop it in a tissue processing cassette and then to your ethanol dehydrating steps.

Jerry Ricks
Research Scientist
University of Washington
Department of Pathology





> Date: Wed, 12 May 2010 06:50:14 -0700
> From: kmerriam2003 <@t> yahoo.com
> To: histonet <@t> lists.utsouthwestern.edu
> Subject: [Histonet] FFPE cell pellet prep
> 
> Sorry - I forgot to put a subject line.
> 
> 
> Good morning,
> 
> I am trying to make some nice FFPE cell pellet blocks, but I seem to lose a lot of cells along the way (especially when trying to take the cells out of the tube).  We are fixing the cells in NBF, spinning them down, adding 70% and spinning down again.  At that point, I am trying to scoop out the cells (with a weighing scoop) and wrap them in lens paper for processing.  I am losing a lot of the cells during this step, because the pellet is not quite solid.  Do you think it would be OK to let the cells air-dry a bit and then take them out for processing?  I know this goes against everything I was taught in histology, but I am really at a loss here.
> 
> Anyone have any hot tips for me?
> 
> Kim
>  Kim Merriam, MA, HT(ASCP)QIHC
> Cambridge, MA 
> 
> 
> 
>       
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