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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
' _% a5 v# |4 {( u' m1. Remove embryos (E13–E16) from a pregnant mouse, rinse in PBS, remove and discard
4 U& F/ [! c+ j' v& Q8 Eplacenta and fetal membranes, head, liver and heart. Retain and rinse the carcasses in PBS.
! x1 }* J/ z& U. Y5 _; C2. 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
' ]8 K* J! t' j0 ?1 f6 Hmagnetic 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,7 q% t% a) b7 h' z1 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).
7 i; m1 {8 y; C) T' W3 G3 L! u3. 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
O1 d! C [) JMEF growth medium and centrifuge at 450g for 5 min at RT. Resuspend the pellet in about 3 ml of MEF growth medium.
" f; U$ F2 T% ZMEF growth medium: MEF growth medium DMEM (4.5 g/ liter glucose) supplemented with 15%
* G7 k! W$ X$ r4 I9 u' P(vol/vol) FBS and 1%penicillin/streptomycin solution for cultivation of MEFs.Store at 4 ℃ for up to 2 weeks.& z) ?: k9 {1 g/ Y6 F/ F! Z* G
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
4 L: O3 \3 z& F0 |$ p/ v. d1 yMEF growth medium at 5% CO2 and 37℃ for 24 h./ o& j6 W$ t( k' g* Z
5. After 24 h, change the medium to remove debris, erythrocytes and unattached cellular aggregates.
( U- G- C: S' f" |. A6. Cultivate for an additional 1–2 d until the cells reach B90% confluence.! L9 a1 c, |! K: |
7. Rinse the MEF plate with Ca2+- and Mg2+-free PBS twice.
' ^" @) F0 g# a+ L8 T" o/ U. T8. Add trypsin solution to the culture plates and incubate for 1–2 min." @( D& ~* o6 L. A3 c
9. Aspirate trypsin solution, collect cells in MEF growth medium and expand them once (1:3 split).
) G: [% `$ m8 k$ Z1 u10. Freeze any MEFs not needed immediately.: U# d/ N; N4 d; @# P# n
-POINT MEFs at P0 can be stored in liquid nitrogen up to 1 year. . q; R! Y; [* B; s6 O' d
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
! m3 p# f9 q: O/ Uundifferentiated maGSCs; prepare as follows.
7 Y1 s/ I/ e2 Q7 W( D3 f) l+ p$ d12. Incubate a confluent plate of MEFs with medium containing mitomycin C (10 ug/ ml) at 37 ℃ for 3 h.. x6 d) x& t! H' M, B9 ~; |
13. Aspirate the mitomycin C solution and wash three times with PBS.
5 S+ ?! O& j1 |9 n) k14. 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
0 i" k% {9 p! }. Q6 b% X+ }. C0 Jat a density of 50,000–60,000 cells/ cm2.7 T! [3 _+ M# z: z- K
-CRITICAL STEP MEFs prepared 1 d before maGSC subculture are optimal. Although established cell lines may grow well on 2- to& z7 o8 X7 D2 q) {5 i& z
3-d-old MEFs, it is critical to use MEFs within 1 d after mitomycin C treatment for primary cultures (early derivation steps, mechanical
- ]* H" h4 P" u! A+ Apassaging and thawing). |
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发表于 2010-5-7 04:20
发表于 2011-7-26 19:21
