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本帖最后由 qgjin 于 2010-5-7 04:30 编辑 9 f, f5 F8 N& e
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PREPARATION OF MEF CULTURES
" ]0 r2 K5 |) u, b% u, [/ v1. Remove embryos (E13–E16) from a pregnant mouse, rinse in PBS, remove and discard
A" b( ^& |$ l2 G1 U: z) iplacenta and fetal membranes, head, liver and heart. Retain and rinse the carcasses in PBS.) U4 w' b6 A4 `: J# W8 J- j
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& l9 N* R3 s/ Q5 O$ F% Z- D
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,
% N2 v1 |9 b2 @' }7 ]( _$ w% tthe resulting cell suspension should be essentially free of any larger pieces of tissue and should not be too viscous (genomic DNA-lysed cells)." Q2 B) |8 C* Y4 C* b5 `
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/ Z; j4 M5 Y( q. V
MEF growth medium and centrifuge at 450g for 5 min at RT. Resuspend the pellet in about 3 ml of MEF growth medium.& i" T9 G7 ~' Y8 V/ Q7 T
MEF growth medium: MEF growth medium DMEM (4.5 g/ liter glucose) supplemented with 15%
# \1 Q) V1 G3 T7 R(vol/vol) FBS and 1%penicillin/streptomycin solution for cultivation of MEFs.Store at 4 ℃ for up to 2 weeks.
9 t, A2 D9 _8 v3 |# x6 D" w4. 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% ^5 e- P3 \. A3 r, \# [9 }4 R
MEF growth medium at 5% CO2 and 37℃ for 24 h.5 ]. S0 ?# `6 H2 \: R
5. After 24 h, change the medium to remove debris, erythrocytes and unattached cellular aggregates.
' m$ C# B2 t% p. d' @( r6. Cultivate for an additional 1–2 d until the cells reach B90% confluence.
/ C! c6 |6 Q8 C3 w: l$ Y7. Rinse the MEF plate with Ca2+- and Mg2+-free PBS twice.
$ D9 Z ?# Y: c7 [8. Add trypsin solution to the culture plates and incubate for 1–2 min.
# @% p1 h4 J2 B! `9. Aspirate trypsin solution, collect cells in MEF growth medium and expand them once (1:3 split).9 I! B, x% I& f7 O' _
10. Freeze any MEFs not needed immediately.1 i0 h) v8 b N+ U9 G
-POINT MEFs at P0 can be stored in liquid nitrogen up to 1 year. : F8 f/ M# I+ }. y& B% g3 R
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& P0 J& H8 T( ^3 z5 L
undifferentiated maGSCs; prepare as follows.
# u7 K7 U3 L( i12. Incubate a confluent plate of MEFs with medium containing mitomycin C (10 ug/ ml) at 37 ℃ for 3 h.
5 i- B9 K* q: Y& n& q5 L, P) _3 P2 {13. Aspirate the mitomycin C solution and wash three times with PBS.6 Z# n% R5 n4 }. y5 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# L3 R- N- y9 B5 v
at a density of 50,000–60,000 cells/ cm2.
/ }0 c' m& k' ?, }7 i" S -CRITICAL STEP MEFs prepared 1 d before maGSC subculture are optimal. Although established cell lines may grow well on 2- to
& y% G& ^3 {% z, }7 L) X6 a2 [* v3-d-old MEFs, it is critical to use MEFs within 1 d after mitomycin C treatment for primary cultures (early derivation steps, mechanical5 l% t- P6 K; ~
passaging and thawing). |
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发表于 2010-5-7 04:20
发表于 2011-7-26 19:21
