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本帖最后由 qgjin 于 2010-5-7 04:30 编辑
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9 F; K) ^0 ]$ \7 }PREPARATION OF MEF CULTURES% l6 v: h7 B4 u
1. Remove embryos (E13–E16) from a pregnant mouse, rinse in PBS, remove and discard
# U! u6 z7 C2 k8 F1 Tplacenta and fetal membranes, head, liver and heart. Retain and rinse the carcasses in PBS.
% K/ b* S0 [" V% s, Q6 g2. 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
- B& X0 H) W7 J7 L5 \* V/ Lmagnetic 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,, H$ b4 M, C5 e8 K$ L7 B
the resulting cell suspension should be essentially free of any larger pieces of tissue and should not be too viscous (genomic DNA-lysed cells).
4 \5 Q/ f2 f1 c. D) `7 O6 D. g, O3. 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
3 k! W& b8 H# q5 wMEF growth medium and centrifuge at 450g for 5 min at RT. Resuspend the pellet in about 3 ml of MEF growth medium.
: y& D4 T5 W8 U2 C, HMEF growth medium: MEF growth medium DMEM (4.5 g/ liter glucose) supplemented with 15%
" `- V- M5 B; s: M N/ H(vol/vol) FBS and 1%penicillin/streptomycin solution for cultivation of MEFs.Store at 4 ℃ for up to 2 weeks.
+ ~+ c) r( \# f8 _6 B2 y y4. 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 in0 J1 R g& ^2 t" c7 Y( Q
MEF growth medium at 5% CO2 and 37℃ for 24 h.
! d2 J3 ]1 ?9 o/ u5. After 24 h, change the medium to remove debris, erythrocytes and unattached cellular aggregates.
5 u6 }% r' u. z& v( M& m6. Cultivate for an additional 1–2 d until the cells reach B90% confluence.
" n/ X8 I7 i' t; e& B7. Rinse the MEF plate with Ca2+- and Mg2+-free PBS twice./ }( Y: {8 A+ S
8. Add trypsin solution to the culture plates and incubate for 1–2 min./ z9 U: {3 p5 U M' N
9. Aspirate trypsin solution, collect cells in MEF growth medium and expand them once (1:3 split).
! p$ R% q- L( ~- p10. Freeze any MEFs not needed immediately.
( ^' O! |* j+ P5 }+ c2 r-POINT MEFs at P0 can be stored in liquid nitrogen up to 1 year.
: V' T5 s$ T9 }) ~* Y11. 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& d/ N: v: J7 ]+ e0 P g" D" J6 U# q" N! e
undifferentiated maGSCs; prepare as follows.4 c, C8 J' a& R$ m
12. Incubate a confluent plate of MEFs with medium containing mitomycin C (10 ug/ ml) at 37 ℃ for 3 h.
$ c6 x7 H* Z& {. [: C13. Aspirate the mitomycin C solution and wash three times with PBS.# p; h' Y7 w, M9 J0 R2 f9 N: E
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
6 w$ x% T$ l8 G; a7 m1 j& \at a density of 50,000–60,000 cells/ cm2.4 X W5 T( Y+ g5 Z; l" r8 Y+ g) m8 c
-CRITICAL STEP MEFs prepared 1 d before maGSC subculture are optimal. Although established cell lines may grow well on 2- to4 m! A' X) [, j, t
3-d-old MEFs, it is critical to use MEFs within 1 d after mitomycin C treatment for primary cultures (early derivation steps, mechanical0 [2 a6 t- A/ a4 C/ {! ~! f! V
passaging and thawing). |
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发表于 2010-5-7 04:20
发表于 2011-7-26 19:21
