[Histonet] RE: Embedding cells in Paraffin

[James Watson]

We do this all the time.  Here is our protocol:

Agarose Pre-embedding Cell Cultures Pellets Collected in 15 ml. Tubes

Material:

•	10% Neutral Buffer Formalin
•	1X Dulbeccos PBS 
•	NuSieve GTG Low Melting Point Agarose: Lonza, Cat# 50080
•	Corning 15 ml conical Centrifuge tubes 

Solutions:

1% Neutral Buffered Formalin
		10% Neutral Buffered Formalin……………………………………………….10 ml
		1X DPBS………………………………………..............................................90 ml

	3.0% Agarose
		NuSieve Low Melting Point Agarose powder…………………….……3.0 gm
	1% Neutral Buffered Formalin………………………………….…..……100.0 ml
1.	In a fume hood, warm up the mixture until the agarose completely dissolves and maintain the solution at approximately 40C.
2.	Pipette 1 ml of liquid agarose gel into the 1.5 ml micro-centrifuge tubes. 
3.	Let solidify and store at room temperature.



Procedure for Making Cultured Cell Pellets

1.	Culture cells in desired number of vessels (e.g. 3 T-175 flasks) so that you will have 40-60 million cells at appropriate confluence.
2.	Cell harvesting: take out part of culture medium and leave 5-10 ml of culture medium in each flask. With the flask placed on ice, quickly scrape off cells using a sterile cell scraper.
3.	Spin down the cells at 4°C, 1000 rpm, 5 min. Resuspend cells in cold PBS and then combine cell suspensions from different flasks into one 15 ml tube. (If the total volume of detached cells from all flasks is less than 15 ml, directly transfer cell suspensions to one 15 ml tube.
4.	Spin at 1500 rpm, 4°C, for 20 minutes.
5.	Carefully discard supernatant without disturbing cell pellet.
6.	Along the tube wall, carefully add 4 ml of 10% Neutral Buffered Formalin (NBF) into each tube and incubate at room temperature overnight.
7.	Discard 10% NBF without disrupting cell pellet.
8.	Carefully add 0.5 ml of 70% ETOH along the tube wall and hold overnight at room temperature. Samples can be stored in 70% ETOH at 4°C at this step for up to 3 days before proceeding.
9.	Warm the 3% Agarose to 40°C until completely melted.
10.	Remove as much of the 70% ETOH as possible.
11.	Pipette on 1.0 ml of the melted Agarose onto the washed cultured cells.
12.	Spin down the tube immediately in a centrifuge at 1000-1500 rpm for 2-5min.
13.	Make sure that the cultured cells are at the bottom of the tube.
14.	Carefully cut the tube slightly above the top of the cell pellet, carefully release the cell pellet into a 35 x 10 mm Tissue Culture Dish containing 3% NuSieve LMP Agarose solution maintained at approximately 40°C. 
15.	Once agarose completely solidified, bring the pellets to the Histology lab for paraffin embedding.




Procedure for processing Cultured Cell Pellets (Histology)

1.	Add one or two drops of 70% ETOH to side of well plate to loosen pellet from wall of well.
2.	Carefully pop agarose using the modified spatula and wrap in lens paper.
3.	Process the pellet on the normal mouse cycle on the tissue processor using the 100% ETOH without the 5% glycerin.
4.	Embed the pellet with the cultured cells at the bottom of the mold.


James Watson HT  ASCP
GNF  Genomics Institute of the Novartis Research Foundation
Tel    858-332-4647
Fax   858-812-1915
jwatson <@t> gnf.org

-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu [mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Mike Tighe
Sent: Monday, December 02, 2013 9:06 AM
To: histonet <@t> lists.utsouthwestern.edu (histonet <@t> lists.utsouthwestern.edu)
Subject: [Histonet] Embedding cells in Paraffin

Has anyone tried to embed cells grown in tissue culture? I am trying to put some tissue culture cells through same stress as tissue would go through. Fixation, dehydration, and heat. Any ideas? I could re-suspend in OCT and then fix for extended time with NBF but that doesn't quite seem fair.



Thanks for any ideas!
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