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
  G7 l& C& n) P7 S1 R; }Isolation of Primary Fibroblasts from Mouse Embryos0 i, P' l) p  a

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( m7 i5 t+ f$ z% g" G/ ~) ?0 \" o        1.        Treat 10 cm tissue culture plates with 0.1% gelatin
: v: S- Z8 K* _1 e+ U! }for at least two hours before use.
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  o7 f8 U' ~: p$ w- @3 r        2.        Sacrifice the pregnant female mouse (day 13 or 14 p.c.)
+ [# \- j8 u, u5 L* G* b, {/ j8 eby cervical dislocation.  Dissect out the uterine horns and place into
# Z/ P: E' G9 ?a petri dish containing PBS.
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! g# h$ Y% N$ M0 ]5 R8 d$ W        3.        Separate each embryo from its placenta and surrounding
& U4 R2 S& t3 L7 C8 n- n6 D" rmembranes.  If desired, keep the yolk sac for genotyping.' q" [7 K* f* v8 [- ?9 q' ^) e

9 l9 f9 m5 ^' `9 R( l! |( D        4.        Wash each embryo by transferring it to a petri dish containing
! m  o( x3 X! l1 U! Iclean PBS.# m/ Q. h7 c, \' N9 n
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        5.        Using a Pipetman with the end of the tip snipped off, & w1 r3 r: r0 A* Q5 V0 E" K
transfer the embryo into the barrel of a 1cc syringe fitted with an 8 X, i3 P( w" e7 C4 g
18G 11/2" needle and, using the syringe's plunger, force the embryo * U" a4 z; k2 ?" w( z: M
through the needle into a sterile 15 ml tube containing 1 ml of medium
  Q" z- v0 t7 F2 i(DMEM, 1X GPS, and 15% FCS).
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        6.        Working in the laminar flow hood, aseptically transfer the
6 Z- F4 e7 [' t6 \- \( R1 ml of embryo cells to a sterile 15 ml tube containing 10 ml of medium / P& N) u! E/ L+ O; i0 K5 i
(DMEM, 1X GPS, and 15% FCS).
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; p" p* c9 z) v. `9 k+ J        7.        Aspirate the gelatin from the 10 cm plates and plate out the
( n& C. w  m  Z4 a3 \suspension of embryonic cells.2 @0 c3 @4 ]) Q& R- N
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        8.        Incubate the plates @ 37o C until the cells have reached   Z& W' G# [+ V& w: J
confluence (approx. 5 days).  (The primary fibroblasts will be the only
  }: H( X4 D+ L8 {9 v/ n4 pcells that attach and proliferate.)6 G- G2 |1 G1 q; i$ g0 D! q

+ s6 p  O5 X% E& \' \- l0 T        9.        At confluence, split the plate 1:3 by doing the following:
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, C; z' O/ l1 A. R        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 5 m! l' p1 ]/ n- R0 O6 }/ j2 v
            for 7 minutes.
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        c.        Add 5 ml of medium (DMEM, 1X GPS, and 10% FCS) to the 5 c/ b5 D" |+ B5 A2 w1 |% H; N
            plate to neutralize the trypsin.  Resuspend the cells by
9 w/ T  e$ s. C8 [$ B" S2 A            pipetting up-and-down several times.
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, ]" C' M6 s* ~        d.        Uniformly divide the cells among three 10 cm gelled plates, 7 O5 n. n7 U8 D4 _4 K( i
            each containing 8 ml of medium (DMEM, 1X GPS, and 10% FCS).  5 R& B$ ^& l; f$ c" X$ p' f
            These plates are designated as Passage No. 1.
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1 h" D! n: J( s4 ^, X2 F, ?        10.        Upon confluence, harvest and freeze the primary fibroblasts 1 f2 M/ C0 F, x' h- R
@ a freezing density of 3.0 x 107 cells/ml.  Reserve a fraction of the
: J1 s' _. U# C2 A$ c$ ~cells to seed a 6 cm gelled dish from which DNA will be prepared for
: D# L5 _8 e( V. Agenotyping (unless the yolk sac was used for genotypic analyis).  
# e$ j4 v+ a3 b/ E" b$ V& S5 {The frozen cells are designated as Passage No. 2.
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, g! ?2 b- Y2 d: [# j) IFrom the Laboratory of Dr. Allan Bradley                      
# J4 N- d4 x% s: V; gBaylor College of Medicine, Houston, Texas

anql

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mominglily

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