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本帖最后由 qgjin 于 2010-5-7 04:30 编辑 ( ^; X- x. b+ \' f0 ]. V2 h4 W
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PREPARATION OF MEF CULTURES& T$ e k0 S/ h( Q' ?+ {8 q
1. Remove embryos (E13–E16) from a pregnant mouse, rinse in PBS, remove and discard
$ U3 V n# ~7 O. s4 U6 W2 nplacenta and fetal membranes, head, liver and heart. Retain and rinse the carcasses in PBS.
; R, r' ~8 W; ~9 k8 |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
) k+ W# n: ~& Y: b3 t) Pmagnetic 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,
2 n# Z& h" S% |( |) h- f+ x8 A6 pthe resulting cell suspension should be essentially free of any larger pieces of tissue and should not be too viscous (genomic DNA-lysed cells).
" Z5 l# y' R6 K; H7 x3. 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
- w& g K( R. x6 c6 ^# d7 zMEF growth medium and centrifuge at 450g for 5 min at RT. Resuspend the pellet in about 3 ml of MEF growth medium.
: A# D4 [ p C; W2 zMEF growth medium: MEF growth medium DMEM (4.5 g/ liter glucose) supplemented with 15%
% H* M% y3 h8 e3 w% J5 A(vol/vol) FBS and 1%penicillin/streptomycin solution for cultivation of MEFs.Store at 4 ℃ for up to 2 weeks.
, N6 s F, ~. ]* C6 y4. 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
' T' n* U2 U- qMEF growth medium at 5% CO2 and 37℃ for 24 h.4 h: I& z9 P4 i$ v8 p3 G6 Q
5. After 24 h, change the medium to remove debris, erythrocytes and unattached cellular aggregates.6 b9 ?$ P( v& \
6. Cultivate for an additional 1–2 d until the cells reach B90% confluence.
' o# @! ^0 u# `: c8 g- ~7. Rinse the MEF plate with Ca2+- and Mg2+-free PBS twice.( z( ~/ O6 a$ B3 j# _
8. Add trypsin solution to the culture plates and incubate for 1–2 min.
0 b# }8 F6 ^+ O7 d; p/ ~9. Aspirate trypsin solution, collect cells in MEF growth medium and expand them once (1:3 split).
8 D4 b8 ]" [' N6 q: t/ ~# Y6 f% ?10. Freeze any MEFs not needed immediately.
% }# b: b. }+ b& }* T6 e$ X* ^9 Y-POINT MEFs at P0 can be stored in liquid nitrogen up to 1 year.
' ?% q# Y- ?5 ^( T11. 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
# N# K# @( Y8 k! `/ q, o _undifferentiated maGSCs; prepare as follows.
$ Q! ^+ f) H4 F _( G1 g; t12. Incubate a confluent plate of MEFs with medium containing mitomycin C (10 ug/ ml) at 37 ℃ for 3 h., k5 z8 Y7 A+ s' r( B( }
13. Aspirate the mitomycin C solution and wash three times with PBS.
8 i4 h+ Q3 H7 M j14. 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+ j/ D" s- @6 X9 M9 }# h, T- p
at a density of 50,000–60,000 cells/ cm2.. g5 ?0 w+ x) ]' {' d& r% M. q
-CRITICAL STEP MEFs prepared 1 d before maGSC subculture are optimal. Although established cell lines may grow well on 2- to
! t- d5 J8 @! B) r. R! X$ X% L$ @3-d-old MEFs, it is critical to use MEFs within 1 d after mitomycin C treatment for primary cultures (early derivation steps, mechanical5 G$ w" F3 H* @- Z0 C' S0 A" Q4 C
passaging and thawing). |
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发表于 2010-5-7 04:20
发表于 2011-7-26 19:21
