Isolation of Primary Fibroblasts from Mouse Embryos 从小鼠胚胎分离纤维原细胞Bayl

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Isolation of Primary Fibroblasts from Mouse Embryos Allan Bradley's Lab, Baylor College of Medicine, Houston, Texas 从小鼠胚胎分离纤维原细胞
; |% |1 D4 z+ q  H& O- V$ l! aIsolation of Primary Fibroblasts from Mouse Embryos
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        1.        Treat 10 cm tissue culture plates with 0.1% gelatin / B# e. v7 Z1 F; J$ ^
for at least two hours before use.4 s5 Y2 W; |$ r

8 n- H& W: ^/ s5 A. y        2.        Sacrifice the pregnant female mouse (day 13 or 14 p.c.) # {& r9 G9 I5 \4 P5 i8 L2 Y
by cervical dislocation.  Dissect out the uterine horns and place into ; h0 `; ]# M1 \7 w2 a! K
a petri dish containing PBS.
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        3.        Separate each embryo from its placenta and surrounding 5 r/ b* v6 v! ]# o- ~9 l# g
membranes.  If desired, keep the yolk sac for genotyping./ V# z1 ^1 ^% l9 y+ i( X9 w
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        4.        Wash each embryo by transferring it to a petri dish containing & x5 g# F. P! M
clean PBS.  p# \( w. A% S$ C+ a4 |
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        5.        Using a Pipetman with the end of the tip snipped off,
4 k6 W) Y+ a" q0 b( K, R1 ltransfer the embryo into the barrel of a 1cc syringe fitted with an % Z. R  u8 y/ ~! @4 d
18G 11/2" needle and, using the syringe's plunger, force the embryo
* E9 E( {; \/ L5 `  ethrough the needle into a sterile 15 ml tube containing 1 ml of medium 2 \8 g& V& P4 V
(DMEM, 1X GPS, and 15% FCS).
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$ M9 H5 o' m5 b) K1 `. R        6.        Working in the laminar flow hood, aseptically transfer the % T6 Y. s! \. H" A- L+ q- w
1 ml of embryo cells to a sterile 15 ml tube containing 10 ml of medium
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        7.        Aspirate the gelatin from the 10 cm plates and plate out the
* w% m# [- p2 ?0 s) r! o; x6 R( Vsuspension of embryonic cells.
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        8.        Incubate the plates @ 37o C until the cells have reached ) N1 t' U2 a! ~2 |' R) Z
confluence (approx. 5 days).  (The primary fibroblasts will be the only
/ l3 H( x& ^3 I5 ncells that attach and proliferate.)
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% V3 K/ f3 y7 F, o4 \! c) U        9.        At confluence, split the plate 1:3 by doing the following:6 ^2 H3 v1 k+ B) R( V3 N+ Y

( G: a% S( d3 e0 Z9 r" I' K4 N        a.        Wash the plate twice with PBS.
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        b.        Add 2 ml of trypsin per 10 cm dish and incubate @ 37o C
; o; }1 [) u3 M/ z4 `            for 7 minutes.
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        c.        Add 5 ml of medium (DMEM, 1X GPS, and 10% FCS) to the
6 x3 x; Q6 @2 D4 A- }2 i+ T8 y( {! {            plate to neutralize the trypsin.  Resuspend the cells by 1 F  }+ x: h' c6 o& P( ^& U
            pipetting up-and-down several times.
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' i- X: r- ?( ~6 H7 [        d.        Uniformly divide the cells among three 10 cm gelled plates, * N( T1 d) S  j
            each containing 8 ml of medium (DMEM, 1X GPS, and 10% FCS).  
8 N) {2 k5 w. Z& R, I2 T            These plates are designated as Passage No. 1.4 r) S2 @/ P$ T% I3 \& D
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        10.        Upon confluence, harvest and freeze the primary fibroblasts
9 f1 Z, V0 j$ h: J; I( ?5 |2 U8 F  j& X@ a freezing density of 3.0 x 107 cells/ml.  Reserve a fraction of the
  j1 Y$ E- a! k; m# kcells to seed a 6 cm gelled dish from which DNA will be prepared for
  ^! p1 Y4 K/ L8 r5 rgenotyping (unless the yolk sac was used for genotypic analyis).  
9 Z2 m5 R/ T5 h* B# V2 f7 T  P5 FThe frozen cells are designated as Passage No. 2.. N  c, H$ \; h; p3 o. @! Z& d; w& H

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From the Laboratory of Dr. Allan Bradley                       0 A* I3 f- f5 @7 ^6 ~0 Y2 E
Baylor College of Medicine, Houston, Texas

anql

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mominglily

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