|
 
- 积分
- 302
- 威望
- 302
- 包包
- 311
|
本帖最后由 qgjin 于 2010-5-7 04:30 编辑 ! V7 ^5 Y) g; e5 l8 ~' L
. w- q8 `; R1 V+ g% oPREPARATION OF MEF CULTURES. `* T9 I5 W7 k: x' p9 |& _
1. Remove embryos (E13–E16) from a pregnant mouse, rinse in PBS, remove and discard
; A+ y; {+ p7 l) c/ o) } Q- nplacenta and fetal membranes, head, liver and heart. Retain and rinse the carcasses in PBS.
; f( i1 W+ \0 H* }# ~, M! t2. 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
Z$ T( |( i2 [9 `1 ?5 Zmagnetic 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,$ o1 j$ Q9 A! m( K
the resulting cell suspension should be essentially free of any larger pieces of tissue and should not be too viscous (genomic DNA-lysed cells).* Q7 m8 p# @( h+ |( H$ @
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& t$ O2 z; ], e3 n' [8 _+ I
MEF growth medium and centrifuge at 450g for 5 min at RT. Resuspend the pellet in about 3 ml of MEF growth medium.; \2 e& y' V& J
MEF growth medium: MEF growth medium DMEM (4.5 g/ liter glucose) supplemented with 15%
; i6 `0 m1 ~3 g2 G* J$ ]* k(vol/vol) FBS and 1%penicillin/streptomycin solution for cultivation of MEFs.Store at 4 ℃ for up to 2 weeks.1 b& D0 a! A- r1 c
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 E G! `2 W, y f p0 e8 z1 V
MEF growth medium at 5% CO2 and 37℃ for 24 h.2 p9 ]) |2 U$ B) E
5. After 24 h, change the medium to remove debris, erythrocytes and unattached cellular aggregates.
# G% Q' Q2 k7 J4 q$ _' d3 J6. Cultivate for an additional 1–2 d until the cells reach B90% confluence.
. ?0 a' V# J0 A. Z r( Q7. Rinse the MEF plate with Ca2+- and Mg2+-free PBS twice.
/ z% K! u& e5 q; j( |6 O8. Add trypsin solution to the culture plates and incubate for 1–2 min.4 G: A) O% p& |7 x
9. Aspirate trypsin solution, collect cells in MEF growth medium and expand them once (1:3 split).1 B6 r! s. I: _8 l
10. Freeze any MEFs not needed immediately." u$ S0 v) j, _, ]7 p5 |
-POINT MEFs at P0 can be stored in liquid nitrogen up to 1 year. + c; |: j8 h t& B6 ~% M$ [
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
4 z0 B, _- ~% t* ?/ m1 bundifferentiated maGSCs; prepare as follows.
7 b1 u# K- H/ |: Z" K9 H$ E" B' s12. Incubate a confluent plate of MEFs with medium containing mitomycin C (10 ug/ ml) at 37 ℃ for 3 h.
/ I$ M6 ?# S( V: S' o. V13. Aspirate the mitomycin C solution and wash three times with PBS.
5 V* _1 [# u/ i* a4 X14. 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
. N8 R+ h$ `# U% Zat a density of 50,000–60,000 cells/ cm2.& N) i' x6 _7 J' u- n( a( i0 ?& g
-CRITICAL STEP MEFs prepared 1 d before maGSC subculture are optimal. Although established cell lines may grow well on 2- to
6 o- C2 `' l6 Q! n( a3-d-old MEFs, it is critical to use MEFs within 1 d after mitomycin C treatment for primary cultures (early derivation steps, mechanical; |( @* M$ @9 Z' D9 f- s
passaging 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
