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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 g _4 k1 l" l1 s. A
1. Remove embryos (E13–E16) from a pregnant mouse, rinse in PBS, remove and discard
. L0 M% @) w8 q; t! U/ h- I: Gplacenta and fetal membranes, head, liver and heart. Retain and rinse the carcasses in PBS.! M7 n ?7 {; U
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
' \) Y, _7 Q! S5 _/ 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,! f9 Q, b$ c3 C4 e7 w9 e
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 N( I' M2 c3 e$ u i6 f# E |/ V" ]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
! k% `2 p" @' d4 f |" U2 W7 D+ rMEF growth medium and centrifuge at 450g for 5 min at RT. Resuspend the pellet in about 3 ml of MEF growth medium.# O+ }9 W) j: Q/ K& i2 y
MEF growth medium: MEF growth medium DMEM (4.5 g/ liter glucose) supplemented with 15%6 ~' f# k& J; W0 ?8 P3 z
(vol/vol) FBS and 1%penicillin/streptomycin solution for cultivation of MEFs.Store at 4 ℃ for up to 2 weeks.
`+ t6 j1 l: Y1 `& K4. 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 C9 m, r% G' t# {$ [
MEF growth medium at 5% CO2 and 37℃ for 24 h.2 R; Y" d6 i3 Q8 e7 [8 y2 d% W
5. After 24 h, change the medium to remove debris, erythrocytes and unattached cellular aggregates.
/ u, }9 U# q; t! U2 ?0 V3 f( E5 W+ O7 Z6. Cultivate for an additional 1–2 d until the cells reach B90% confluence. m' w7 }! [8 @. P. j0 Q/ ]
7. Rinse the MEF plate with Ca2+- and Mg2+-free PBS twice.$ c, w/ ?$ i# M/ g" o
8. Add trypsin solution to the culture plates and incubate for 1–2 min." i6 \, A' W j
9. Aspirate trypsin solution, collect cells in MEF growth medium and expand them once (1:3 split).. o1 Y' s* e( S' @2 x& a8 K! @
10. Freeze any MEFs not needed immediately., P3 v n# ~: Q4 v2 X+ m
-POINT MEFs at P0 can be stored in liquid nitrogen up to 1 year.
; o, ?# G9 ^. g7 r5 j* ^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 of
; S9 }6 `* E' V7 q" u- @5 z6 h1 K9 fundifferentiated maGSCs; prepare as follows.
2 o2 Y/ X) s; U0 @" D- e12. Incubate a confluent plate of MEFs with medium containing mitomycin C (10 ug/ ml) at 37 ℃ for 3 h.3 t4 |, a, P" G% N7 S7 u" u( R6 v
13. Aspirate the mitomycin C solution and wash three times with PBS.1 j# o% 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
, d- D. a. p9 wat a density of 50,000–60,000 cells/ cm2.1 E$ _) y7 P9 U8 ^- R
-CRITICAL STEP MEFs prepared 1 d before maGSC subculture are optimal. Although established cell lines may grow well on 2- to" f2 M' ?0 W! B2 h! W( p
3-d-old MEFs, it is critical to use MEFs within 1 d after mitomycin C treatment for primary cultures (early derivation steps, mechanical
- ^- B8 Z M) a9 {' ~passaging and thawing). |
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发表于 2010-5-7 04:20
发表于 2011-7-26 19:21
