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本帖最后由 qgjin 于 2010-5-7 04:30 编辑 ! P- N2 G) C& }. X& R4 E4 o
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PREPARATION OF MEF CULTURES
3 J* {$ H! m' o! @4 J1. Remove embryos (E13–E16) from a pregnant mouse, rinse in PBS, remove and discard
3 h+ p# Z5 f) F. {7 Eplacenta and fetal membranes, head, liver and heart. Retain and rinse the carcasses in PBS.
/ z- e8 J! h! D$ b: Y2. 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
* o9 F, z/ }5 Q. V$ cmagnetic 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,
/ }" p) t$ | C7 d0 i1 x; dthe resulting cell suspension should be essentially free of any larger pieces of tissue and should not be too viscous (genomic DNA-lysed cells).
3 h! ^- L9 N( U- `( d3. 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
/ m Y: \5 J3 X, S5 PMEF growth medium and centrifuge at 450g for 5 min at RT. Resuspend the pellet in about 3 ml of MEF growth medium." ]: _% s. T. D
MEF growth medium: MEF growth medium DMEM (4.5 g/ liter glucose) supplemented with 15%# ~. j% [) G. v5 y* y2 {1 g8 n
(vol/vol) FBS and 1%penicillin/streptomycin solution for cultivation of MEFs.Store at 4 ℃ for up to 2 weeks.
2 \! ]% g. O$ E4 J4. 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: {- U+ a3 B# K( W8 G/ l
MEF growth medium at 5% CO2 and 37℃ for 24 h.1 U3 n+ {5 {1 Q
5. After 24 h, change the medium to remove debris, erythrocytes and unattached cellular aggregates.8 h0 \& h; @/ J* \+ S2 L# Y: N( P
6. Cultivate for an additional 1–2 d until the cells reach B90% confluence.' m4 A/ V+ {0 s5 f) @
7. Rinse the MEF plate with Ca2+- and Mg2+-free PBS twice.( r Q- R' q6 A8 G
8. Add trypsin solution to the culture plates and incubate for 1–2 min.3 b! O! [3 b2 {# d8 P1 ~
9. Aspirate trypsin solution, collect cells in MEF growth medium and expand them once (1:3 split).
, Y7 U+ i( F3 j- }# ^10. Freeze any MEFs not needed immediately.9 g+ s" T _4 |+ a
-POINT MEFs at P0 can be stored in liquid nitrogen up to 1 year. , w- Z: m- D1 I9 v$ z+ c' J
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
( |3 C2 t6 Q& ^9 k- _4 d4 Dundifferentiated maGSCs; prepare as follows.) Y# j* T* v! F
12. Incubate a confluent plate of MEFs with medium containing mitomycin C (10 ug/ ml) at 37 ℃ for 3 h.) e3 {0 M3 v" T' r& _* ~1 X
13. Aspirate the mitomycin C solution and wash three times with PBS., c9 W- c! X7 m9 N: d0 j- T
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 dishes0 R! ^+ M3 @$ J, I9 H
at a density of 50,000–60,000 cells/ cm2." J" H. l. ~" @ F; z( S
-CRITICAL STEP MEFs prepared 1 d before maGSC subculture are optimal. Although established cell lines may grow well on 2- to$ _5 V% W& _- f6 V! i- K
3-d-old MEFs, it is critical to use MEFs within 1 d after mitomycin C treatment for primary cultures (early derivation steps, mechanical3 @6 X# D( ~" N6 W w% E
passaging and thawing). |
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发表于 2010-5-7 04:20
发表于 2011-7-26 19:21
