Isolation of Primary Fibroblasts from Mouse Embryos





	1.	Treat 10 cm tissue culture plates with 0.1% gelatin 

for at least two hours before use.



	2.	Sacrifice the pregnant female mouse (day 13 or 14 p.c.) 

by cervical dislocation.  Dissect out the uterine horns and place into 

a petri dish containing PBS.



	3.	Separate each embryo from its placenta and surrounding 

membranes.  If desired, keep the yolk sac for genotyping.



	4.	Wash each embryo by transferring it to a petri dish containing 

clean PBS.



	5.	Using a Pipetman with the end of the tip snipped off, 

transfer the embryo into the barrel of a 1cc syringe fitted with an 

18G 11/2" needle and, using the syringe's plunger, force the embryo 

through the needle into a sterile 15 ml tube containing 1 ml of medium 

(DMEM, 1X GPS, and 15% FCS).



	6.	Working in the laminar flow hood, aseptically transfer the 

1 ml of embryo cells to a sterile 15 ml tube containing 10 ml of medium 

(DMEM, 1X GPS, and 15% FCS).



	7.	Aspirate the gelatin from the 10 cm plates and plate out the 

suspension of embryonic cells.



	8.	Incubate the plates @ 37o C until the cells have reached 

confluence (approx. 5 days).  (The primary fibroblasts will be the only 

cells that attach and proliferate.)



	9.	At confluence, split the plate 1:3 by doing the following:



	a.	Wash the plate twice with PBS.



	b.	Add 2 ml of trypsin per 10 cm dish and incubate @ 37o C 

            for 7 minutes.



	c.	Add 5 ml of medium (DMEM, 1X GPS, and 10% FCS) to the 

            plate to neutralize the trypsin.  Resuspend the cells by 

            pipetting up-and-down several times.



	d.	Uniformly divide the cells among three 10 cm gelled plates, 

            each containing 8 ml of medium (DMEM, 1X GPS, and 10% FCS).  

            These plates are designated as Passage No. 1.



	10.	Upon confluence, harvest and freeze the primary fibroblasts 

@ a freezing density of 3.0 x 107 cells/ml.  Reserve a fraction of the 

cells to seed a 6 cm gelled dish from which DNA will be prepared for 

genotyping (unless the yolk sac was used for genotypic analyis).  

The frozen cells are designated as Passage No. 2.