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本帖最后由 qgjin 于 2010-5-7 04:30 编辑 & L+ C$ U8 y. W+ C7 m& Y! B
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PREPARATION OF MEF CULTURES+ f( `/ i, g# u3 Z e/ c3 F
1. Remove embryos (E13–E16) from a pregnant mouse, rinse in PBS, remove and discard, D# H* n! d& p! j" d/ j
placenta and fetal membranes, head, liver and heart. Retain and rinse the carcasses in PBS.
7 j V' w0 L4 |7 X2. 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
9 `( @ c2 e2 R! ~/ X; i/ 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,
5 q+ s$ Y t7 u8 D9 ?6 H7 Fthe resulting cell suspension should be essentially free of any larger pieces of tissue and should not be too viscous (genomic DNA-lysed cells).
* A% X: T6 Z6 V, }/ n3. 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 of6 f) q& h+ N! ~3 o; ~5 ?
MEF growth medium and centrifuge at 450g for 5 min at RT. Resuspend the pellet in about 3 ml of MEF growth medium.6 }1 b0 [3 e; I6 V) B
MEF growth medium: MEF growth medium DMEM (4.5 g/ liter glucose) supplemented with 15%
4 {, w {' B# J(vol/vol) FBS and 1%penicillin/streptomycin solution for cultivation of MEFs.Store at 4 ℃ for up to 2 weeks.+ ]9 f* m$ {7 m( E
4. 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 in$ S& X2 N" `; `
MEF growth medium at 5% CO2 and 37℃ for 24 h.+ c0 r9 l1 o7 U6 k5 ^/ h' l. h
5. After 24 h, change the medium to remove debris, erythrocytes and unattached cellular aggregates.! M9 b" U* w- o' U
6. Cultivate for an additional 1–2 d until the cells reach B90% confluence.7 i- w0 W: R# o$ Y8 L
7. Rinse the MEF plate with Ca2+- and Mg2+-free PBS twice.1 [/ M- E; \: ^) W
8. Add trypsin solution to the culture plates and incubate for 1–2 min. r3 [; F3 X8 g, i, P
9. Aspirate trypsin solution, collect cells in MEF growth medium and expand them once (1:3 split).9 B! R! s/ L' q, r% Z
10. Freeze any MEFs not needed immediately.
# L. q/ x# x; i: Q; P) V# ]( R" J-POINT MEFs at P0 can be stored in liquid nitrogen up to 1 year. ! v4 m0 f6 l& m: A* k: f: m4 Y
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- M% {- x% m+ U8 a6 E/ G+ h1 u
undifferentiated maGSCs; prepare as follows.7 }3 F1 b* w" A b
12. Incubate a confluent plate of MEFs with medium containing mitomycin C (10 ug/ ml) at 37 ℃ for 3 h.( X. {9 b9 J' x$ K) p( m' K6 v
13. Aspirate the mitomycin C solution and wash three times with PBS.
7 k; `- g( M: u9 z0 }14. 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 dishes. C% F1 |7 [0 {" e" e5 Y" b
at a density of 50,000–60,000 cells/ cm2.% {9 T1 C: u0 c& D3 B
-CRITICAL STEP MEFs prepared 1 d before maGSC subculture are optimal. Although established cell lines may grow well on 2- to
) {& m: e+ ]2 u3 c7 k3-d-old MEFs, it is critical to use MEFs within 1 d after mitomycin C treatment for primary cultures (early derivation steps, mechanical1 ^# ]0 ^9 L# _3 O, h
passaging and thawing). |
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发表于 2010-5-7 04:20
发表于 2011-7-26 19:21
