|
 
- 积分
- 302
- 威望
- 302
- 包包
- 311
|
本帖最后由 qgjin 于 2010-5-7 04:30 编辑 + b0 Y3 e5 l" a O9 N5 E u. q
+ p2 {9 N4 L4 I' B
PREPARATION OF MEF CULTURES6 `4 {5 o# R/ |6 z* J
1. Remove embryos (E13–E16) from a pregnant mouse, rinse in PBS, remove and discard
8 B+ W; `5 h0 z% {% mplacenta and fetal membranes, head, liver and heart. Retain and rinse the carcasses in PBS. p; n3 E! w9 Y/ I( M/ |' e* I& Z
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
& n" U# a$ D* L$ ~2 M9 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, }1 M# m2 \1 z
the resulting cell suspension should be essentially free of any larger pieces of tissue and should not be too viscous (genomic DNA-lysed cells).
+ k+ ~. G, q( I0 y3. 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) p: M8 D3 ]& e) \
MEF growth medium and centrifuge at 450g for 5 min at RT. Resuspend the pellet in about 3 ml of MEF growth medium.
& ^9 P" S9 ~( V8 gMEF growth medium: MEF growth medium DMEM (4.5 g/ liter glucose) supplemented with 15%
3 q9 j8 e2 Z) ?5 H/ _, ]: q; s(vol/vol) FBS and 1%penicillin/streptomycin solution for cultivation of MEFs.Store at 4 ℃ for up to 2 weeks.
) P* E- s( j0 @8 f1 n+ U( p4. 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
/ }1 z4 k J! T7 v( n; o9 n1 e% GMEF growth medium at 5% CO2 and 37℃ for 24 h.( Y4 _0 T) Z' k- u
5. After 24 h, change the medium to remove debris, erythrocytes and unattached cellular aggregates.$ r# k$ t& ]2 J5 k2 w2 W/ n
6. Cultivate for an additional 1–2 d until the cells reach B90% confluence.
" n% l9 P- M. ?$ f7. Rinse the MEF plate with Ca2+- and Mg2+-free PBS twice.& z( h% d' a' L9 S
8. Add trypsin solution to the culture plates and incubate for 1–2 min.: {3 l* o2 ?1 f" x4 n s9 ]# t4 D
9. Aspirate trypsin solution, collect cells in MEF growth medium and expand them once (1:3 split).9 l8 A2 v; V6 Y% @
10. Freeze any MEFs not needed immediately.) i. k J8 O' |9 d. P' H
-POINT MEFs at P0 can be stored in liquid nitrogen up to 1 year. ( E+ @, E- X- S) m( M3 S: S, o
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 of4 k6 [) y8 D) t3 A! d6 ~
undifferentiated maGSCs; prepare as follows.
2 O) b; n- N. a( V+ H% t4 J' ?9 y12. Incubate a confluent plate of MEFs with medium containing mitomycin C (10 ug/ ml) at 37 ℃ for 3 h.
! `( f# e1 c6 \. |+ m( E6 F13. Aspirate the mitomycin C solution and wash three times with PBS.
* v' }; T i- B' N14. 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
* T8 ~' ]$ n- I, Dat a density of 50,000–60,000 cells/ cm2.* H# E# W9 b: f' b) ^2 a3 g
-CRITICAL STEP MEFs prepared 1 d before maGSC subculture are optimal. Although established cell lines may grow well on 2- to" |0 x& n' Z" J
3-d-old MEFs, it is critical to use MEFs within 1 d after mitomycin C treatment for primary cultures (early derivation steps, mechanical
8 l8 T, N7 D$ [2 l- Bpassaging and thawing). |
-
总评分: 威望 + 5
包包 + 10
查看全部评分
|
发表于 2010-5-7 04:20
发表于 2011-7-26 19:21
