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: kmerriam2...@yahoo.com > To: histonet@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 > > > > > _______________________________________________ > Histonet mailing list > Histonet@lists.utsouthwestern.edu > http://lists.utsouthwestern.edu/mailman/listinfo/histonet _________________________________________________________________ The New Busy is not the old busy. Search, chat and e-mail from your inbox. http://www.windowslive.com/campaign/thenewbusy?ocid=PID28326::T:WLMTAGL:ON:WL:en-US:WM_HMP:042010_3_______________________________________________ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
