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本帖最后由 qgjin 于 2010-5-7 04:30 编辑
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5 n# C/ V4 y5 ~0 v" l$ [9 k2 x- ?PREPARATION OF MEF CULTURES
5 z3 j8 Z4 A" f) K7 G! [1. Remove embryos (E13–E16) from a pregnant mouse, rinse in PBS, remove and discard3 V F4 G9 L& @0 v
placenta and fetal membranes, head, liver and heart. Retain and rinse the carcasses in PBS.9 o) q4 b6 E. _ q+ u5 [
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
1 d* T# o! ~/ K" c& fmagnetic 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,
0 ]7 e- Q( D2 }0 R3 F4 ~0 T8 _the resulting cell suspension should be essentially free of any larger pieces of tissue and should not be too viscous (genomic DNA-lysed cells).1 J8 {9 P C4 ?4 ]( Y& \
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
# g5 F x! h. G1 \MEF growth medium and centrifuge at 450g for 5 min at RT. Resuspend the pellet in about 3 ml of MEF growth medium.4 l+ h; F6 x4 {% l- H! z
MEF growth medium: MEF growth medium DMEM (4.5 g/ liter glucose) supplemented with 15%( s. J4 z+ L. [( j
(vol/vol) FBS and 1%penicillin/streptomycin solution for cultivation of MEFs.Store at 4 ℃ for up to 2 weeks.4 i/ |* N2 Z; B% c8 T' X- ^* D' r
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, p. s: d0 G, H$ q$ z: {
MEF growth medium at 5% CO2 and 37℃ for 24 h.! M3 }6 T: N0 Y2 K8 k. @
5. After 24 h, change the medium to remove debris, erythrocytes and unattached cellular aggregates.& _7 H+ m5 u+ g8 a, B2 `
6. Cultivate for an additional 1–2 d until the cells reach B90% confluence.
! f) o4 @. T; A, ]" M2 X6 m% c* @7. Rinse the MEF plate with Ca2+- and Mg2+-free PBS twice.8 x( y0 b8 w+ m. @ K+ D' _
8. Add trypsin solution to the culture plates and incubate for 1–2 min.
+ ~& f# C8 v3 u4 U( ?9. Aspirate trypsin solution, collect cells in MEF growth medium and expand them once (1:3 split).
7 U$ h7 P; U( j% {, x10. Freeze any MEFs not needed immediately.' h1 {4 ]$ ?! v3 V
-POINT MEFs at P0 can be stored in liquid nitrogen up to 1 year.
: g/ _$ E" p( ^* e/ X9 f' a11. 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
/ [; g! N0 J' }) \& y2 O( gundifferentiated maGSCs; prepare as follows.
5 X3 |) [9 l6 L* C2 c1 c8 a12. Incubate a confluent plate of MEFs with medium containing mitomycin C (10 ug/ ml) at 37 ℃ for 3 h.
4 \0 Q4 v" R! V+ ~, L- R' I1 A+ ]13. Aspirate the mitomycin C solution and wash three times with PBS. q' o! J8 k+ ^2 W1 x
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: b+ _/ _" Iat a density of 50,000–60,000 cells/ cm2.4 w ?5 f/ J: I$ u8 a
-CRITICAL STEP MEFs prepared 1 d before maGSC subculture are optimal. Although established cell lines may grow well on 2- to
' H% `7 t. b) ~8 J3 _& W3-d-old MEFs, it is critical to use MEFs within 1 d after mitomycin C treatment for primary cultures (early derivation steps, mechanical8 u9 ?" u5 E/ Y R8 f. m
passaging and thawing). |
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发表于 2010-5-7 04:20
发表于 2011-7-26 19:21
