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本帖最后由 qgjin 于 2010-5-7 04:30 编辑 ( K1 s0 `; e- ]4 w7 p
& d- U8 N, c: Z4 ]3 mPREPARATION OF MEF CULTURES9 K5 [- J4 X$ U3 _
1. Remove embryos (E13–E16) from a pregnant mouse, rinse in PBS, remove and discard
* E" e" Z! ^/ k) yplacenta and fetal membranes, head, liver and heart. Retain and rinse the carcasses in PBS.6 S( u/ ?& j% c% G4 D4 ?
2. Mince the embryonic tissue in 5 ml of trypsin solution, transfer to an Erlenmeyer flask containing a stir bar and a few 5mm glass beads, and stir on
0 v8 L. R, A! O1 a1 z- a: N# \) ^magnetic stirrer for 15–25 min (use shorter incubation times if the embryos are E13 and use longer incubation times if the embryos are E16). Ideally,0 d+ B [$ L; u! ]( k$ K
the resulting cell suspension should be essentially free of any larger pieces of tissue and should not be too viscous (genomic DNA-lysed cells).1 y. f3 f* V* _* ^ U7 S: L; r* _
3. Pipette the cells with a 2-ml glass pipette to achieve cell suspension. Filter the suspension through a sieve or a screen, add 10 ml of7 ?1 m( z) Z' M; O9 M" m8 V
MEF growth medium and centrifuge at 450g for 5 min at RT. Resuspend the pellet in about 3 ml of MEF growth medium.) s/ k* s4 G+ M" O: |
MEF growth medium: MEF growth medium DMEM (4.5 g/ liter glucose) supplemented with 15%2 _( e9 o6 q9 K9 v* R
(vol/vol) FBS and 1%penicillin/streptomycin solution for cultivation of MEFs.Store at 4 ℃ for up to 2 weeks.
9 e! o8 d/ A4 L3 R4. Plate the cell suspension onto cell culture plates at a density of about 2*106 cells per 100 mm plate (P0; i.e., passage 0) and incubate in2 P7 G0 o3 b$ d& S; `$ j5 }! _
MEF growth medium at 5% CO2 and 37℃ for 24 h.# j0 o' x' k! k
5. After 24 h, change the medium to remove debris, erythrocytes and unattached cellular aggregates., K9 y& e+ @5 W% k: M
6. Cultivate for an additional 1–2 d until the cells reach B90% confluence.! G# A8 |; C- J
7. Rinse the MEF plate with Ca2+- and Mg2+-free PBS twice.
4 p. `$ s* n: e0 }, U( ?9 ], b: l8. Add trypsin solution to the culture plates and incubate for 1–2 min.2 m* p4 P& o$ ]% K( g1 M r# }
9. Aspirate trypsin solution, collect cells in MEF growth medium and expand them once (1:3 split).
6 j+ K5 K$ }# L7 K4 N10. Freeze any MEFs not needed immediately.
# m5 h; ^0 O: P6 A. P5 |1 p-POINT MEFs at P0 can be stored in liquid nitrogen up to 1 year.
& |5 \3 [& R) P" V5 k: _11. Continue to passage cells as described in Steps 6–9 of this Box. MEFs at passages 2–4 are most suitable as feeder layer for cultivation of; L0 z- _& d* [' w4 f$ w) c
undifferentiated maGSCs; prepare as follows.+ y& m6 F3 l5 M* I' o. k" H1 m
12. Incubate a confluent plate of MEFs with medium containing mitomycin C (10 ug/ ml) at 37 ℃ for 3 h.$ t5 q5 p5 N$ y4 G; X* L
13. Aspirate the mitomycin C solution and wash three times with PBS.
: v( r. J; v! c# o. h. Q14. Trypsinize MEFs as described in Steps 7–9 of this Box and replate them on new gelatin (0.1%)-treated microwell plates or to Petri dishes1 K6 |: S3 @1 D4 a
at a density of 50,000–60,000 cells/ cm2." {; r; `7 Z* P3 n2 O- i
-CRITICAL STEP MEFs prepared 1 d before maGSC subculture are optimal. Although established cell lines may grow well on 2- to8 _8 [' L# h% B! X% g* K. v* V
3-d-old MEFs, it is critical to use MEFs within 1 d after mitomycin C treatment for primary cultures (early derivation steps, mechanical& q3 M, [1 \% }4 U% g; T
passaging and thawing). |
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发表于 2010-5-7 04:20
发表于 2011-7-26 19:21
