乳腺癌干细胞原代培养问题····大侠们进来帮帮忙

小木梅

关于做原代的时候，组织消化酶和用量，时间的问题5 f8 i- w$ v" G1 G* K) ]* {) j2 |3 y
1.有些文献用胶原酶/透明质酸酶，有些用胶原酶Ⅲ，有的用胶原酶Ⅱ和Ⅳ。。。。请问用什么酶有什么讲究吗？很疑惑哎) [6 n& V+ l2 h# A( u
2.需不需要去除血细胞这一步？, k# p5 V6 a  m/ P/ |- o
3.麻烦有经验的大侠贴个protocol出来参考一下下啊……传授一点经验吧
+ z' Y8 X  q) q6 r+ W- l0 u7 U3 v/ q0 ]
相当疑惑啊。。。

小木梅

amosummer

Stock
9 N( c( n9 X4 H. A! r  e5 mconcentration
) M0 A/ n* d' C. u(mg/ml)
9 s7 i1 \3 U! ~; r3 V& ]) n, uWorking; Z/ K- `2 w! ]8 p
concentration
4 v" B5 o- a6 Q( U9 L7 s(mg/ml)
' H5 j% @3 K+ v6 }2 uVolume( S5 U- ^* n0 P# n* P# {
(ml)5 }/ v* Z& o$ H  H- f8 K+ \/ M. V9 ^7 I
Collagenase 200 0.4 0.1
$ L' V- G4 S) x/ j/ \# x, X& I4 iDispase 200 0.5 0.125
2 g9 m! i, J7 ~+ t& c* ?9 [! sDNase I 100 0.2 0.1
. y4 ^+ m( S: p( V: P0 U" mDulbecco’s phosphatebuffered' Y( V# C& x# ^. {
saline (DPBS)
2 A6 h+ d# T1 w( f- r49.6758 d9 s% U  R- y2 D+ N" {
Collagenase, dispase, DNase I stock . Store at −20°C. Stocks can be
% K) f, t: e2 U- r3 I5 |: T9 v4 `) a6 {made according to manufacturer’s instructions.
+ Q) }1 F3 c6 R! oEnzyme mixture solution: Make fresh as required, store
0 X3 q: M' R2 {9 z" p3 Dat 4°C.% G( I' j9 T7 Q; z8 z, x9 H$ l
2. Materials0 R; W$ Y6 G7 ?" o
2.1. Preparation of. B! i+ q1 K3 X6 Z6 f  c/ Z- t
Cancer Samples
6 |& B- Q+ P5 w; i6 z. i% x* j2.2. Dissociation of  V  Y3 I6 P1 @7 O2 Y$ C  Q2 v
Primary Cancer into
4 f5 T6 f) i6 XSingle Cells
: D* k, d- f4 l( r1 J& _; e2.2.1. Equipment

amosummer

3.2. Dissociation of Primary Cancer into Single Cells
' i; a1 R# b/ b: {1. Transfer an extracted glioblastoma sample into 100-mm 29 y5 A- C* C4 e8 x9 E6 t
culture dish and then wash the sample several times with icecold7 F) n/ j# I( u7 Z) @8 f3 p
DPBS. While washing the sample, remove blood cloths
* c1 v1 H  h# O& H( Q  R0 q( Yand gross necroses.
. ?$ m* M0 }$ u; X# _+ F" q1 D: U2. Divide the sample into 1-cm 3 blocks using a scissor and
( O: }0 @! p$ i1 }- ], K2 l8 stransfer them into 1.5-ml E-tubes. Samples should be in' M- d  P. ], s2 R. }6 @
ice-cold DPBS during the procedure to prevent tissues from
6 ]% ?7 K8 N, f7 Y" a$ ~5 Bbeing dried.& p* F- a  p: q! W: {
3. Dissociate the blocks mechanically by chopping them many
2 k7 k1 }2 Z; `# Z7 v+ `times with scissors as shown in Fig. 3a .
3 D+ C, C5 M& I: p4 {4. After primary mincing, collect the samples in a 15-ml conical5 `& d+ S8 _/ v% c" |3 b
tube or a 1.5-ml E-tube and triturate them further by sucking. I9 a4 n% r$ ]4 q0 m4 _7 M
them in a Pasteur pipette multiple times as shown in Fig. 3 b# Y6 F# ]9 q2 r9 D& }$ A1 n
( see Note 2 . for air bubbles).( c& _+ c1 l5 r2 f
5. Wash the sample several times with ice-cold DPBS./ u( I2 B# W* S1 V3 b7 k) X, W# @5 V
6. Digest the sample using the enzyme mixture solution ( see; p+ ]+ E# U6 D$ l' z
Note 3 . for alternative enzymes). The ratio of enzyme mixture
6 ^# K% b6 P1 ^% U6 ]- fsolution and mechanically dissociated sample volume is
8 y  L1 |+ m$ yusually 1:1. The enzymatic digestion is persisted for 30–60
4 ?4 A2 e$ C% k' c3 Rmin at 37°C. After the first 30 min of incubation, mechanical5 E+ K  @8 U+ f) i+ c7 D& F& D
mixing or mild trituration can be applied.5 `% ]6 m) _: I, t4 R3 |1 ^8 g
7. Wash the chemically digested sample several times with icecold9 y( n2 l6 u5 J! c3 _, G5 [8 G
DPBS./ g5 p3 ]9 l% K0 M
8. Filter the mechanically and chemically dissociated sample" q3 v8 [7 \1 p  {( a
through a cell strainer (40 μ m pore size) and collect single
' e# G) Z' r  ]% F! ucancer cells as shown in Fig. 4 .! x9 s  F: m$ n" c* P$ _
9. Collected cancer cells can be contaminated by red blood7 X) l) F6 F$ R  ]
cells (RBCs). To eliminate RBCs, carefully layer the cancer. ^, t# U( l) C9 p
cell suspension on 15 ml Ficoll-Paque PLUS (1.077 density)$ K7 n  c! d3 u
in a 50-ml conical tube.: [1 L* j0 \4 E* O6 F# B/ G
10. Centrifuge it at 400 × g (2,000 rpm) for 20 min at room
. V3 K% h, K* Htemperature without brake.& j7 d$ ?! J' y, O/ F2 N
11. Take the cancer cell layer (middle) between DPBS (upper)
2 G% z. r' I* Z- i. p' C: band Ficoll-Paque PLUS (lower) using a Pasteur pipette.
7 v9 K8 ~8 [" p( P12. Wash the cancer cells with ice-cold DPBS for several times.& p9 K, {' k9 n( E, c/ X
13. If cell debris still remains, allow the cancer cell suspension to9 L% r# \& X9 ?6 e
stand for over 15 min at 4°C and remove 70% of upper portion* g' V- r( N9 c+ r3 ^8 h' s: _' O
of suspension containing unwanted cell debris.% h8 e6 G  e* S: z
14. Count cancer cell number. Counting dead cells can be
; l, Z) u- _9 O9 _avoided by using Trypan blue staining.

amosummer

3.2. Dissociation of Primary Cancer into Single Cells
1 |4 L& Z# n6 j  ]! q1. Transfer an extracted glioblastoma sample into 100-mm 2' e2 z4 M& ]1 K; w4 r5 p$ `; c
culture dish and then wash the sample several times with icecold
9 j' P: A) ~) _  F( W% B0 LDPBS. While washing the sample, remove blood cloths
$ S% p; ^+ n4 _- B3 A. ?0 x2 Cand gross necroses.
) z: r3 y; G% g& h2. Divide the sample into 1-cm 3 blocks using a scissor and& D& \: c$ j2 }& _  ]
transfer them into 1.5-ml E-tubes. Samples should be in5 t. p& X4 u) w: n4 V! g6 o
ice-cold DPBS during the procedure to prevent tissues from$ q' X7 [: D: n) g" ?
being dried.
( o& y9 ~4 M! z3 [: O; v* O2 {3. Dissociate the blocks mechanically by chopping them many/ H) O- }+ N$ q2 N+ }, Z- E
times with scissors as shown in Fig. 3a .
/ ?) c0 e& j' r' _4. After primary mincing, collect the samples in a 15-ml conical
0 u) Y% G: [; m! ?  H0 k+ @& ]3 _tube or a 1.5-ml E-tube and triturate them further by sucking1 C: R. l0 P6 G' J( X2 N8 H1 Y7 d' c
them in a Pasteur pipette multiple times as shown in Fig. 3 b
) a# `  W# q$ ~  p7 H! O% s; d( see Note 2 . for air bubbles).
1 y5 g/ ^2 m! ~6 h  i5. Wash the sample several times with ice-cold DPBS.4 W6 V$ ~# |  ]% E# k
6. Digest the sample using the enzyme mixture solution ( see
( z9 }4 M# ^& O5 T5 ?Note 3 . for alternative enzymes). The ratio of enzyme mixture* X2 `. R. D% q3 i! j
solution and mechanically dissociated sample volume is
- @! w( v' y: F5 J5 g% X, Jusually 1:1. The enzymatic digestion is persisted for 30–60
6 n6 S: U0 o( emin at 37°C. After the first 30 min of incubation, mechanical1 r8 S" g& |! C# k
mixing or mild trituration can be applied.
7 Y3 A+ `. c2 |! R2 a* t7. Wash the chemically digested sample several times with icecold
- r. B1 q6 g- o" @5 A4 B, I7 cDPBS.! h& g) R/ h$ g
8. Filter the mechanically and chemically dissociated sample. J2 w9 T9 Y6 z8 k7 n' o
through a cell strainer (40 μ m pore size) and collect single
; B1 }2 }1 S7 @' P% I- ccancer cells as shown in Fig. 4 .8 D- [0 J! t9 W! s
9. Collected cancer cells can be contaminated by red blood# m. {: L4 i2 j/ I3 S; L+ S3 r
cells (RBCs). To eliminate RBCs, carefully layer the cancer
; Z2 r* d$ V# x9 s( ocell suspension on 15 ml Ficoll-Paque PLUS (1.077 density)0 D0 t0 W8 U1 W9 }! Q+ i, e2 T) p- E; D
in a 50-ml conical tube.
$ s) h. E" S/ C8 y10. Centrifuge it at 400 × g (2,000 rpm) for 20 min at room5 {" E, C! }. \' O( z
temperature without brake.
. l' Z6 P3 d- _0 A11. Take the cancer cell layer (middle) between DPBS (upper)' C$ B3 e% y2 ^) H" V. j6 B/ j- I& a
and Ficoll-Paque PLUS (lower) using a Pasteur pipette.2 L+ S$ f2 f/ T+ n
12. Wash the cancer cells with ice-cold DPBS for several times.
; z% W- \- E- \6 t5 v/ i13. If cell debris still remains, allow the cancer cell suspension to5 p$ o  y3 W6 A9 u- R! |
stand for over 15 min at 4°C and remove 70% of upper portion
0 J6 G" [3 Z! Y# Pof suspension containing unwanted cell debris.
% y6 K- h: t" I# Q' a: u4 `; P14. Count cancer cell number. Counting dead cells can be
. M; L% o. }& x& Uavoided by using Trypan blue staining.

pzypig

回复 amosummer 的帖子9 J, {" V, W2 c. t) o" ?1 S

$ K( D2 L' V/ b1 v请问您回复的是什么，来源于何处呢，可否提供，谢谢

amosummer

回复 pzypig 的帖子6 v9 O9 }* y1 \# F- k2 ?2 `# u
% @+ I# P) h" a; b6 I
论坛里面的电子书cancer stem cell protocol

hzxyq

好啊