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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 o$ _. T: h5 |/ S' |
1. Remove embryos (E13–E16) from a pregnant mouse, rinse in PBS, remove and discard
0 ?) F7 @/ L6 G) j# U7 \8 z- Uplacenta and fetal membranes, head, liver and heart. Retain and rinse the carcasses in PBS.( `, Y8 {. ?9 d6 S5 W3 W
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; R. \3 o" V) Z1 @- D: p
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,
- I7 r' ?4 Z( U) X9 O+ ithe resulting cell suspension should be essentially free of any larger pieces of tissue and should not be too viscous (genomic DNA-lysed cells). c+ S8 I$ E+ }: `
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
6 ~4 N5 g; @# G* H9 l$ {MEF growth medium and centrifuge at 450g for 5 min at RT. Resuspend the pellet in about 3 ml of MEF growth medium.) `/ I8 v' p. f" D
MEF growth medium: MEF growth medium DMEM (4.5 g/ liter glucose) supplemented with 15%
0 Z( t$ L' V5 D) F4 Q(vol/vol) FBS and 1%penicillin/streptomycin solution for cultivation of MEFs.Store at 4 ℃ for up to 2 weeks.
# K6 ?/ ]4 o' y0 v4 g$ t- S! m4. 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
0 [* B& C; z0 b! HMEF growth medium at 5% CO2 and 37℃ for 24 h./ }6 z; D& E- V( x6 Y4 c
5. After 24 h, change the medium to remove debris, erythrocytes and unattached cellular aggregates.
4 K4 C1 b' k( |) \- m) I$ S) W6. Cultivate for an additional 1–2 d until the cells reach B90% confluence.
6 X2 `7 u8 F# y) s. F7. Rinse the MEF plate with Ca2+- and Mg2+-free PBS twice.. i+ h3 F: u6 V( k( l7 I
8. Add trypsin solution to the culture plates and incubate for 1–2 min.+ {) ]# | y6 @* c! c$ [5 w
9. Aspirate trypsin solution, collect cells in MEF growth medium and expand them once (1:3 split).9 K; ^# \0 u& T, M Q) b9 g2 _6 w! i
10. Freeze any MEFs not needed immediately.
8 p. A2 \2 H+ N, {6 J-POINT MEFs at P0 can be stored in liquid nitrogen up to 1 year.
y0 P5 V& \* ^" O) v; R0 G8 N( A. W11. 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$ }0 O* A% D `# b4 S7 M- ~
undifferentiated maGSCs; prepare as follows.
7 I1 X) K# n v! p8 K4 F12. Incubate a confluent plate of MEFs with medium containing mitomycin C (10 ug/ ml) at 37 ℃ for 3 h.
`4 K5 ], z- _+ h: B1 a13. Aspirate the mitomycin C solution and wash three times with PBS.
9 O) O4 s' z, \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
9 B: }4 z5 e2 b. i4 i! D8 eat a density of 50,000–60,000 cells/ cm2.! V: C' p% j5 E: @# @2 n3 q; T
-CRITICAL STEP MEFs prepared 1 d before maGSC subculture are optimal. Although established cell lines may grow well on 2- to( Z# Q/ W5 \" z2 a" B
3-d-old MEFs, it is critical to use MEFs within 1 d after mitomycin C treatment for primary cultures (early derivation steps, mechanical
4 v: C( X) o, t( P* |1 ~passaging and thawing). |
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发表于 2010-5-7 04:20
发表于 2011-7-26 19:21
