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本帖最后由 qgjin 于 2010-5-7 04:30 编辑
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PREPARATION OF MEF CULTURES
% D$ L3 H# B( g9 X7 E7 ?3 K3 E9 G1. Remove embryos (E13–E16) from a pregnant mouse, rinse in PBS, remove and discard* y# ^' e+ u, t( ^
placenta and fetal membranes, head, liver and heart. Retain and rinse the carcasses in PBS.. m! f f5 W! `
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% g7 |9 J& \/ n' B, w; z
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,
1 S6 b. i7 W m# g4 O7 Y& }the resulting cell suspension should be essentially free of any larger pieces of tissue and should not be too viscous (genomic DNA-lysed cells).! _- d. v. q% L3 [. j
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 of
9 P8 G* ~2 x1 y1 G |0 m) k2 r9 _MEF growth medium and centrifuge at 450g for 5 min at RT. Resuspend the pellet in about 3 ml of MEF growth medium.
. G8 e) D# V2 }' SMEF growth medium: MEF growth medium DMEM (4.5 g/ liter glucose) supplemented with 15%% p J J% d4 g
(vol/vol) FBS and 1%penicillin/streptomycin solution for cultivation of MEFs.Store at 4 ℃ for up to 2 weeks.
; h2 Z+ o8 a, R+ c; n( x( f/ t4. 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" C& R2 c( p L- U
MEF growth medium at 5% CO2 and 37℃ for 24 h.& d% K4 K1 l& Q" S5 ~: O+ f' \, s
5. After 24 h, change the medium to remove debris, erythrocytes and unattached cellular aggregates.4 P7 |" K7 p; e- {" f6 E- p
6. Cultivate for an additional 1–2 d until the cells reach B90% confluence.
$ k' W) J8 D3 z$ M( ~7. Rinse the MEF plate with Ca2+- and Mg2+-free PBS twice.
* E3 `* G2 D Y5 A2 Y( I$ M( Y& k8. Add trypsin solution to the culture plates and incubate for 1–2 min.
8 ^- V; u! J0 h( d9. Aspirate trypsin solution, collect cells in MEF growth medium and expand them once (1:3 split).1 N# p( y1 `1 V$ H% {7 ^
10. Freeze any MEFs not needed immediately.4 M5 H5 L) S) V9 d
-POINT MEFs at P0 can be stored in liquid nitrogen up to 1 year.
% A) L5 W" q5 t: G! K X11. 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
( L% }! S8 e8 O/ L6 q+ P# Zundifferentiated maGSCs; prepare as follows.6 P# ^0 m! g$ e) q- W3 j
12. Incubate a confluent plate of MEFs with medium containing mitomycin C (10 ug/ ml) at 37 ℃ for 3 h.# ~8 t- L! H7 H+ q
13. Aspirate the mitomycin C solution and wash three times with PBS.: X/ \. C8 b% w# \
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, o. B! q' H0 Q$ S$ |6 d4 B
at a density of 50,000–60,000 cells/ cm2.
5 `2 ?( X9 g' A* i& x) a -CRITICAL STEP MEFs prepared 1 d before maGSC subculture are optimal. Although established cell lines may grow well on 2- to8 n8 w' k$ ~# T1 h
3-d-old MEFs, it is critical to use MEFs within 1 d after mitomycin C treatment for primary cultures (early derivation steps, mechanical8 h, `0 t/ ^% r* y' S
passaging and thawing). |
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发表于 2010-5-7 04:20
发表于 2011-7-26 19:21
