|
  
- 积分
- 1833
- 威望
- 1833
- 包包
- 7854
|
Chondrogenic media without cytokines (CMwoC)—STOCK: 500 mL bottle of
! F+ k' m$ c" @2 m$ ~9 dhigh-glucose DMEM (GIBCO; catalog # 10569-010) supplemented with: 50 μg/mL
; |2 R3 i2 z5 `7 T! ~0 }l-ascorbic-2-phosphate (Sigma; catalog # A8960; 500 μL of 50 mg/mL stock in DI
( N- a' m- L; Y/ x( Z6 Kwater); 40 μg/mL l-proline (Sigma; catalog # P0380; 500 μL of 40 mg/mL stock in
( U+ r. W5 H, ?& O- i! V! yDI water); 100 μg/mL sodium pyruvate (Sigma; catalog # P5280; 500 μL of 100 mg/0 z: m& o- F; q: t; N
mL stock in DI water); 5 mL ITS+ Culture Supplement that consists of 6.25 μg/mL
2 g9 l8 r( \' @9 l' Q0 linsulin, 6.25 μg/mL transferrin, 6.25 ng/mL selenous acid, 1.25 mg/mL bovine serum3 F9 O' ^# y( E |! [
albumin, 5.35 mg/mL linoleic acid (B&D Biosciences; catalog # 35-4352). P# W; y& b0 K2 \1 g
CMwoC can be stored for the duration of the chondrogenic culture at
. b( x% {% w# u3 _0 C, e3 s2–8°C.
4 Y% N* @1 e7 ?5. Chondrogenic media with cytokines (CMwC) – STOCK: Needed volume of
, f* H6 R0 Z' S2 E% }' H& aCMwoC supplemented with: 10 ng/mL rhTGF-β3 (R&D Systems; catalog # 243-B3;
) j! [9 c7 w8 H/ j* U2 o$ tfrom 10 μg/mL stock in 4 mM HCl); 10−7M dexamethasone (Sigma; catalog # D2915,% b; W: h+ M5 A9 |5 ^
100 mg, water soluble; from 1 mM stock in DI water); 500 ng/mL BMP-2 (rhBMP-2,8 r4 S" Y7 y4 k$ x t6 d9 S2 m7 U- |
CHO-derived, R&D Systems, catalog # 355-BM OR rhBMP-6, CHO-derived R&D
6 _1 A( g5 I3 u4 o# u/ cSystems, catalog # 507-BP; from 10 μg/mL stock in PBS, see Note 3).
2 g' g8 a$ w5 c- R- ECMwC should be prepared fresh with every use. Stocks of cytokines should
) V, v5 L7 J# Q$ G+ Wbe aliquoted and frozen at -20 °C to avoid several freeze-thaw cycles, which can
, K/ W8 {* S( z+ J& w! ~inactivate them.
& Y* t: x( T+ ]6 S8 OChondrogenesis Differentiation) V" X! r3 x; F+ G/ a
Harvest cells when 70–80% confluent for this assay. Cells lifted during early to; b7 p1 [! X7 g# P0 h# p0 K
mid-log of growth or those that have reached 100% confluence will not differentiate6 `/ I- U% G7 ^- K/ V5 z; |' r+ a" h
as well, if at all.9 q1 s5 ]% N0 v y8 X
1. Wash harvested MSCs in PBS and resuspend in 1.0 mL CMwC.
8 p+ _- n5 f' P! T! c, T, ]8 @$ w" u2. Do cell count and viability. Adjust to a concentration to 400 viable cells/μL
9 r' _& \- m; Ywith Chondrogenic Media with Cytokines (CMwC).
* R6 N& G& U/ Z, A$ K. qFor example, if cell count of 1 mL of cell suspension gives 1,000,000 cells/mL,) r4 v. @, S {/ V9 v& `/ D
add 1.5 mL CMwC to give 2.5 mL of 400 cells/μL. Thus, 500 μL should contain6 b2 P. t! U- f" t
200,000 cells. i+ x/ ^8 s; F" } O
3. Transfer approx 200,000 MSCs in 500 μL CMwC into a 15-mL conical polypropylene! }$ \ U) l- Z' k
tube./ Y( l, F5 J! d5 P0 e
4. Screw caps on tightly while in hood (sterility is of the utmost importance as no# s3 q% Y2 z4 S
antibiotics are added to the media).
& D$ \ c8 E" x* x5 J5. Centrifuge the 15-mL conical tube at 450 g for 10 min. DO NOT resuspend the. p7 H5 Y8 K" d( M
pellet and DO NOT aspirate the medium.
$ c. Z$ a& o. D {6 V/ s6. Place the conicals into the cell culture incubator, which is humidified at 37 °C
+ Z6 s; I) \* d+ w/ N! Q2 [with 5% CO2. Loosen the caps on the conicals so that they are simply placed
* d0 R1 S8 x( w. won the conicals without screwing on, allowing for full air exchange. Be sure to6 V+ E0 B5 W: r7 m! s
screw the caps on tightly before removing the conicals from the incubator. (see
; p$ q' \" G1 V; m+ G6 L* |Note 7).
2 }4 K9 a; R e" ^7. Pellets should be visible within 24 h.
5 M; u) k% ^2 ~# ?1 s- }, \0 N8. Change media every 3–4 d by using a P-1000 pipet to remove the old media and1 f9 g% {2 J3 d; Q) [, l% t
add fresh CMwC, paying close attention to detach pellet from plastic with each
! v% y. O$ J4 R c0 m9 Fmedia change. Take care not to aspirate the pellet when removing old medium.
# s; d+ @: x m `5 f, {9. At 21 d, chondrocyte pellet should be 2-4 mm in diameter with BMP-2 or8 @! t+ d# l1 p5 `
1–2 mm with BMP-6.
2 l) |4 y- S2 b% U1 b! W5 t6 N7 H10. At 21 d, chondrocyte pellet may be fixed with NBF, embedded in paraffin, cut
/ e7 n" j& \' p1 G5 }3 Ginto 5-μm sections onto slides, and stained with 1% toluidine blue/1% sodium
5 s- |1 l) a( l) c- V; Fborate. (see Note 8).7 m; x$ R6 E' b: t% B) R
具体可参考Mesenchymal Stem Cells Methods and Protocols（P93） k& D6 I+ [- j6 J8 P6 x8 `
PS：你应该有这本电子书，没有的话，论坛里有，呵呵。& t+ a( s& ?. s! O& X* s9 q5 m
祝工作顺利哦 |
-
总评分: 威望 + 5
包包 + 10
查看全部评分
|
发表于 2010-4-29 15:36
发表于 2010-4-29 16:22
发表于 2010-4-30 10:08
