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本帖最后由 qgjin 于 2010-5-7 04:30 编辑
6 R" b$ v( o" F" `, ]! _6 e/ [9 v+ w. ^2 B& h& D" m9 [" O& [: H
PREPARATION OF MEF CULTURES: B# H+ t" X: `6 T+ T
1. Remove embryos (E13–E16) from a pregnant mouse, rinse in PBS, remove and discard
6 d- m& R6 \4 Z( e1 ]placenta and fetal membranes, head, liver and heart. Retain and rinse the carcasses in PBS.1 w- N, w7 ~4 E6 \
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
- e3 ?/ J) \% i, b0 V5 Imagnetic 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 K# y, X$ y7 o- N& R1 k" t
the resulting cell suspension should be essentially free of any larger pieces of tissue and should not be too viscous (genomic DNA-lysed cells).
9 {" @+ ^1 r8 v1 R2 e/ Q3. 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
+ g+ _, Y) T, Y J; y9 D! D+ \9 rMEF growth medium and centrifuge at 450g for 5 min at RT. Resuspend the pellet in about 3 ml of MEF growth medium.
3 ^4 C' ?7 x0 ]8 LMEF growth medium: MEF growth medium DMEM (4.5 g/ liter glucose) supplemented with 15%0 k' F' h3 X+ t5 { a6 C$ B
(vol/vol) FBS and 1%penicillin/streptomycin solution for cultivation of MEFs.Store at 4 ℃ for up to 2 weeks.
}/ F! K* J3 a( C( ^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. P/ z, l" p" _8 j: F% G
MEF growth medium at 5% CO2 and 37℃ for 24 h.7 Q7 g- f3 b- G* w2 {7 r& \
5. After 24 h, change the medium to remove debris, erythrocytes and unattached cellular aggregates.: f/ {! u6 R: a; E9 @; ?9 e& ]
6. Cultivate for an additional 1–2 d until the cells reach B90% confluence.6 j+ M$ v8 S2 i' I) W" `6 [
7. Rinse the MEF plate with Ca2+- and Mg2+-free PBS twice.
, N, _5 K; Y. I3 h0 B- L8. Add trypsin solution to the culture plates and incubate for 1–2 min.
6 ]: y7 O8 M3 K" V& G( w! @. B9. Aspirate trypsin solution, collect cells in MEF growth medium and expand them once (1:3 split).
* \4 s s, a5 E10. Freeze any MEFs not needed immediately.6 |# R, h" W+ ?8 P8 q! B, A& I
-POINT MEFs at P0 can be stored in liquid nitrogen up to 1 year. * e- A" z* A! \6 F
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 of9 q G* \0 A' E. U
undifferentiated maGSCs; prepare as follows.
# f" P3 W0 O$ m. d/ @. y5 [12. Incubate a confluent plate of MEFs with medium containing mitomycin C (10 ug/ ml) at 37 ℃ for 3 h.
) g8 p1 J% j5 i- `13. Aspirate the mitomycin C solution and wash three times with PBS.1 i; Z/ b- Z# G, i" Y
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
' W+ K+ [( O& dat a density of 50,000–60,000 cells/ cm2.
4 x0 h0 c' Q# W I4 x# m8 f/ P -CRITICAL STEP MEFs prepared 1 d before maGSC subculture are optimal. Although established cell lines may grow well on 2- to
7 H7 F2 ^% t# d; _3-d-old MEFs, it is critical to use MEFs within 1 d after mitomycin C treatment for primary cultures (early derivation steps, mechanical: J: a; x% e& R0 ?! Z/ M) ~
passaging and thawing). |
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发表于 2010-5-7 04:20
发表于 2011-7-26 19:21
