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本帖最后由 qgjin 于 2010-5-7 04:30 编辑 / m; r5 P- ]7 ~# M, y
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PREPARATION OF MEF CULTURES
) W: ?5 E# j* z% g& O9 }1. Remove embryos (E13–E16) from a pregnant mouse, rinse in PBS, remove and discard
% U8 M, o- L" M3 \: O! z0 jplacenta and fetal membranes, head, liver and heart. Retain and rinse the carcasses in PBS.5 m; S# ^' Z* n( i( K6 X) g! t8 @
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" \0 M1 w; A5 J" k) ?6 m
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,
) C6 x% S" ]; H" p, C$ y7 Sthe resulting cell suspension should be essentially free of any larger pieces of tissue and should not be too viscous (genomic DNA-lysed cells)." b6 H) Z) O5 q
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% _* J0 h, c: b; }
MEF growth medium and centrifuge at 450g for 5 min at RT. Resuspend the pellet in about 3 ml of MEF growth medium.
# ?# [$ G- a* WMEF growth medium: MEF growth medium DMEM (4.5 g/ liter glucose) supplemented with 15%: R! V! g6 g: E0 C9 B; x# d. l8 ]
(vol/vol) FBS and 1%penicillin/streptomycin solution for cultivation of MEFs.Store at 4 ℃ for up to 2 weeks.8 N- w d0 w/ ^* K5 z; n' Z T
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
3 F" f$ H) h- _7 FMEF growth medium at 5% CO2 and 37℃ for 24 h.
1 e7 H4 z( s# a( |3 [* o5. After 24 h, change the medium to remove debris, erythrocytes and unattached cellular aggregates.9 ^/ Y& B g! @: L# D3 Q$ I
6. Cultivate for an additional 1–2 d until the cells reach B90% confluence.
. Z# {& L3 R1 t5 L7. Rinse the MEF plate with Ca2+- and Mg2+-free PBS twice.0 w( u n' j+ I
8. Add trypsin solution to the culture plates and incubate for 1–2 min.+ p1 m+ f6 }8 _5 O" r
9. Aspirate trypsin solution, collect cells in MEF growth medium and expand them once (1:3 split).. s( M% }8 g0 H
10. Freeze any MEFs not needed immediately.
( F# j% A5 H& X0 H-POINT MEFs at P0 can be stored in liquid nitrogen up to 1 year. & o& `8 A1 h- r# k. g' A& C% }
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' m1 S/ x2 m; m3 Y+ u4 v
undifferentiated maGSCs; prepare as follows.+ _( T% e" _6 }' g/ a7 q% K* ]5 y
12. Incubate a confluent plate of MEFs with medium containing mitomycin C (10 ug/ ml) at 37 ℃ for 3 h.1 S9 `' \. X: J6 A
13. Aspirate the mitomycin C solution and wash three times with PBS.& ?* P u' t0 t$ C: `
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! C; w. B: p- a' F. e/ ~/ n
at a density of 50,000–60,000 cells/ cm2.- ?0 r! M# _8 I- X; X
-CRITICAL STEP MEFs prepared 1 d before maGSC subculture are optimal. Although established cell lines may grow well on 2- to/ w1 s9 f% R/ _" 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
0 i2 T: Z8 A" y5 C" `% j2 cpassaging and thawing). |
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发表于 2010-5-7 04:20
发表于 2011-7-26 19:21
