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本帖最后由 qgjin 于 2010-5-7 04:30 编辑 ' d9 K# i# ^; p/ [; a4 w
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PREPARATION OF MEF CULTURES
6 Z6 R C6 _5 S3 N1. Remove embryos (E13–E16) from a pregnant mouse, rinse in PBS, remove and discard& o4 G+ g' ^' n8 ]5 V
placenta and fetal membranes, head, liver and heart. Retain and rinse the carcasses in PBS.
7 a9 { S2 W }. z4 }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* c& T6 ?& w7 |6 E n
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,
( _! z! ^1 \! T4 p' uthe resulting cell suspension should be essentially free of any larger pieces of tissue and should not be too viscous (genomic DNA-lysed cells).4 X: X- a7 a' o7 U0 e
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) r( a; g+ z) L# L! U3 c0 m2 p5 m
MEF growth medium and centrifuge at 450g for 5 min at RT. Resuspend the pellet in about 3 ml of MEF growth medium.8 g2 J+ j; A. g/ v- Y+ w
MEF growth medium: MEF growth medium DMEM (4.5 g/ liter glucose) supplemented with 15%
3 B/ y3 n$ c. k$ I8 Z/ I(vol/vol) FBS and 1%penicillin/streptomycin solution for cultivation of MEFs.Store at 4 ℃ for up to 2 weeks.& z- \! q' D$ X: P
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 in3 d0 C" Z4 D) H( S
MEF growth medium at 5% CO2 and 37℃ for 24 h.* p. y1 N; J* L# U) Y! z# G3 ~
5. After 24 h, change the medium to remove debris, erythrocytes and unattached cellular aggregates.' }, q$ y! p7 S- p; ?5 n
6. Cultivate for an additional 1–2 d until the cells reach B90% confluence.# G, q, r( N9 z; V& |0 T3 _
7. Rinse the MEF plate with Ca2+- and Mg2+-free PBS twice.
7 v7 q1 U+ y) X3 A7 w0 J8 B: l8. Add trypsin solution to the culture plates and incubate for 1–2 min.
/ ~" O4 ?( Z- A9 e0 ]$ e9. Aspirate trypsin solution, collect cells in MEF growth medium and expand them once (1:3 split).& e3 I0 O, B; ^0 D# q8 c
10. Freeze any MEFs not needed immediately.. U! c$ z* k/ `2 s6 d8 g3 p
-POINT MEFs at P0 can be stored in liquid nitrogen up to 1 year. & k! {+ N; [" @: G' W# O# S
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 of8 J( P1 |( x Q* l. \ d
undifferentiated maGSCs; prepare as follows.$ J8 C& L9 G! f
12. Incubate a confluent plate of MEFs with medium containing mitomycin C (10 ug/ ml) at 37 ℃ for 3 h.
4 ?/ E/ d3 e! ^6 |/ I( ~13. Aspirate the mitomycin C solution and wash three times with PBS.
0 @( ^! V/ T$ D2 g$ j6 `8 g+ [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 dishes6 K! f4 ?' e) K. R) E7 y
at a density of 50,000–60,000 cells/ cm2.7 O2 s: {) C% C; ?& }7 ?. E
-CRITICAL STEP MEFs prepared 1 d before maGSC subculture are optimal. Although established cell lines may grow well on 2- to7 e) s/ S1 @# @- o
3-d-old MEFs, it is critical to use MEFs within 1 d after mitomycin C treatment for primary cultures (early derivation steps, mechanical. H0 V3 H, ?4 p
passaging and thawing). |
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发表于 2010-5-7 04:20
发表于 2011-7-26 19:21
