|
 
- 积分
- 302
- 威望
- 302
- 包包
- 311
|
本帖最后由 qgjin 于 2010-5-7 04:30 编辑 8 [8 k/ K/ a7 K' o
% u! e. ^ e" ~4 B5 {8 ZPREPARATION OF MEF CULTURES i0 v" F5 `( V' l& w4 l. ^ S# }
1. Remove embryos (E13–E16) from a pregnant mouse, rinse in PBS, remove and discard
0 k) f0 A+ i: Z0 \+ \2 aplacenta and fetal membranes, head, liver and heart. Retain and rinse the carcasses in PBS.6 i# }+ i# B- N+ k: D
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
/ _. m8 U( p/ r+ ^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," y! P& D! w1 X! q
the resulting cell suspension should be essentially free of any larger pieces of tissue and should not be too viscous (genomic DNA-lysed cells).
; }) i2 x% ~3 A+ e) O. F0 C3. 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) |8 M3 O3 }; t5 i% R0 G/ N
MEF growth medium and centrifuge at 450g for 5 min at RT. Resuspend the pellet in about 3 ml of MEF growth medium.
& H9 x0 o( U+ V* c: H1 H- \+ E2 SMEF growth medium: MEF growth medium DMEM (4.5 g/ liter glucose) supplemented with 15%2 g- W8 D0 M6 d+ W+ f
(vol/vol) FBS and 1%penicillin/streptomycin solution for cultivation of MEFs.Store at 4 ℃ for up to 2 weeks.
o4 `6 |: x# x% q8 I7 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
) m5 I( i) R _MEF growth medium at 5% CO2 and 37℃ for 24 h.
7 G2 x4 @ _% k9 b0 ^( n/ o5. After 24 h, change the medium to remove debris, erythrocytes and unattached cellular aggregates.
; F. m9 Q* ?' [% i E4 z: {/ \6 t3 A6. Cultivate for an additional 1–2 d until the cells reach B90% confluence.4 @ ]. r* Y/ f7 G6 d
7. Rinse the MEF plate with Ca2+- and Mg2+-free PBS twice.# K X0 j# Q6 S8 B
8. Add trypsin solution to the culture plates and incubate for 1–2 min.
, [8 p/ {+ F- J! j) V S+ _. o5 L9. Aspirate trypsin solution, collect cells in MEF growth medium and expand them once (1:3 split).8 r7 F L/ V: ]* N/ o
10. Freeze any MEFs not needed immediately.
# \1 g3 k( G M. S-POINT MEFs at P0 can be stored in liquid nitrogen up to 1 year. + d3 ^" m9 h5 M, [$ @7 D6 f- 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( `5 X. `% s, \$ s
undifferentiated maGSCs; prepare as follows.
; ~) H$ L2 M9 n- T3 f12. Incubate a confluent plate of MEFs with medium containing mitomycin C (10 ug/ ml) at 37 ℃ for 3 h.
" M1 d, I5 k# D: H, E13. Aspirate the mitomycin C solution and wash three times with PBS." M6 i7 N' l9 }; j3 x! y
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
+ Q& ^6 r; q* [+ f: jat a density of 50,000–60,000 cells/ cm2.
/ J( `- p) E* j2 ] -CRITICAL STEP MEFs prepared 1 d before maGSC subculture are optimal. Although established cell lines may grow well on 2- to: z4 A, a& q+ V, j4 f
3-d-old MEFs, it is critical to use MEFs within 1 d after mitomycin C treatment for primary cultures (early derivation steps, mechanical
: K! i6 X2 P. f- n! Dpassaging 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
