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本帖最后由 qgjin 于 2010-5-7 04:30 编辑 # g2 n7 g" }& H5 i) C
- g& T# W* h4 |# X/ I, M) c* pPREPARATION OF MEF CULTURES! [9 q/ ? U$ B+ S3 @1 g
1. Remove embryos (E13–E16) from a pregnant mouse, rinse in PBS, remove and discard
% P- e9 _+ |/ Y% }placenta and fetal membranes, head, liver and heart. Retain and rinse the carcasses in PBS.
3 |( o' j* S% P7 G" i2. 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% ]! {1 n5 _" _' ~% ?* s8 ? t
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,& V, L) N+ w4 y* T; W8 {5 v, u: c( ~
the resulting cell suspension should be essentially free of any larger pieces of tissue and should not be too viscous (genomic DNA-lysed cells).
. t: A7 p' D7 t" f7 ?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* D& o0 F+ g9 j+ ~
MEF growth medium and centrifuge at 450g for 5 min at RT. Resuspend the pellet in about 3 ml of MEF growth medium.% M0 S" b i. b6 J5 w
MEF growth medium: MEF growth medium DMEM (4.5 g/ liter glucose) supplemented with 15%* k, ]3 J# E8 p& s
(vol/vol) FBS and 1%penicillin/streptomycin solution for cultivation of MEFs.Store at 4 ℃ for up to 2 weeks.
( C5 |% i" k) h3 j+ C5 d+ R4 Q9 O4. 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
7 V& }( E4 C- `. h# cMEF growth medium at 5% CO2 and 37℃ for 24 h.4 i( D/ q) c) }
5. After 24 h, change the medium to remove debris, erythrocytes and unattached cellular aggregates.
6 T5 b1 P0 K& |$ b6. Cultivate for an additional 1–2 d until the cells reach B90% confluence.
4 c9 j7 {/ p) `7. Rinse the MEF plate with Ca2+- and Mg2+-free PBS twice.
, m5 j$ N% w$ ~/ C% k' K q- ^8. Add trypsin solution to the culture plates and incubate for 1–2 min.
2 y7 a& L" v6 v- }" w3 p; [% `9. Aspirate trypsin solution, collect cells in MEF growth medium and expand them once (1:3 split).
5 \) W$ g1 b, e9 M1 V: ]10. Freeze any MEFs not needed immediately.
7 O. |, i0 S( A! J$ I7 O: I* E% n-POINT MEFs at P0 can be stored in liquid nitrogen up to 1 year. + u) u" |$ R$ P! e6 [
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
/ a0 A9 |7 [+ Y# n3 Wundifferentiated maGSCs; prepare as follows.
' R& r; A- m, E' g12. Incubate a confluent plate of MEFs with medium containing mitomycin C (10 ug/ ml) at 37 ℃ for 3 h.
1 _& m' V! W5 ]) F5 v% q i8 x13. Aspirate the mitomycin C solution and wash three times with PBS.
3 j# c) j, @8 L) M& C# ?6 _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
) }5 P- O9 k2 |% Oat a density of 50,000–60,000 cells/ cm2.( C6 J& C) ^9 S! l. {% m
-CRITICAL STEP MEFs prepared 1 d before maGSC subculture are optimal. Although established cell lines may grow well on 2- to
1 x8 Z, c( v4 I5 Q$ [+ k* u3 h3-d-old MEFs, it is critical to use MEFs within 1 d after mitomycin C treatment for primary cultures (early derivation steps, mechanical
. `! g* Y$ a8 b( `% h: {passaging and thawing). |
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发表于 2010-5-7 04:20
发表于 2011-7-26 19:21
