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- 积分
- 1403
- 威望
- 1403
- 包包
- 5968
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3.2. Dissociation of Primary Cancer into Single Cells* }* p( m1 e: z% |
1. Transfer an extracted glioblastoma sample into 100-mm 2
) V2 F, P& u& i$ u6 ^0 Cculture dish and then wash the sample several times with icecold& O s+ |" H7 s0 q: v/ A
DPBS. While washing the sample, remove blood cloths
2 T8 P3 r5 }6 i' I% f( O" B) Z: [1 Band gross necroses.# k0 O" d5 p8 }
2. Divide the sample into 1-cm 3 blocks using a scissor and
R7 v9 R4 Y; T; {4 }! `7 J. w* }transfer them into 1.5-ml E-tubes. Samples should be in
" R: }) z3 T9 M( d" ~9 T3 _ice-cold DPBS during the procedure to prevent tissues from2 V0 B8 z, O' h! G& i
being dried.+ J) Z* D2 x: ]# j& Y( t4 a2 n/ w& k
3. Dissociate the blocks mechanically by chopping them many; B5 z5 b2 C0 D. X, ], W2 Z3 L
times with scissors as shown in Fig. 3a .
9 F, m* d$ R& G' G4. After primary mincing, collect the samples in a 15-ml conical
8 d/ L) j% [9 R2 Z. D& ]tube or a 1.5-ml E-tube and triturate them further by sucking
\/ O) V8 ^8 ythem in a Pasteur pipette multiple times as shown in Fig. 3 b
: }% v# v3 {+ p( i- Q( see Note 2 . for air bubbles).6 z8 z4 X+ o) ~
5. Wash the sample several times with ice-cold DPBS.- K( k2 ]' C( \4 _
6. Digest the sample using the enzyme mixture solution ( see! j+ i- ]+ f6 |# u2 f! x
Note 3 . for alternative enzymes). The ratio of enzyme mixture
; J, ?) z: \8 G) s- Ysolution and mechanically dissociated sample volume is
) ^9 a, [3 x& susually 1:1. The enzymatic digestion is persisted for 30–60# ^! a9 l( _ r1 Q. d4 g+ ?
min at 37°C. After the first 30 min of incubation, mechanical
! V! m. h5 L9 \5 Smixing or mild trituration can be applied.! i6 ~$ A: `! a
7. Wash the chemically digested sample several times with icecold) d2 l# O2 y4 U" Z/ A7 Q2 H8 c
DPBS.- a: Z: ^% N5 |
8. Filter the mechanically and chemically dissociated sample5 m* D5 K t- x; B
through a cell strainer (40 μ m pore size) and collect single$ F5 G- p: q: U# e/ f
cancer cells as shown in Fig. 4 .! L+ z$ |, Y- i2 B; H
9. Collected cancer cells can be contaminated by red blood
8 ?7 `1 u+ y* x" s# Fcells (RBCs). To eliminate RBCs, carefully layer the cancer
# R C; q3 A+ r1 F8 Kcell suspension on 15 ml Ficoll-Paque PLUS (1.077 density)5 ^0 [/ f: r6 L2 v! H
in a 50-ml conical tube.
/ C+ K4 C1 z! {8 Z# l& E+ ]10. Centrifuge it at 400 × g (2,000 rpm) for 20 min at room
9 H. c9 k; p, M, Ytemperature without brake.
: h# ]3 B" v G! |) g d11. Take the cancer cell layer (middle) between DPBS (upper)% m$ v% U( A# j$ Y3 K! n4 X4 x
and Ficoll-Paque PLUS (lower) using a Pasteur pipette.2 l/ q0 S( p* H$ |4 b
12. Wash the cancer cells with ice-cold DPBS for several times.
7 S# k- s9 g* W+ }13. If cell debris still remains, allow the cancer cell suspension to
5 _& x* Z! r3 Y4 rstand for over 15 min at 4°C and remove 70% of upper portion2 }, \, b6 I7 }
of suspension containing unwanted cell debris.1 e, T1 l1 i: ~- F# [5 O2 X& |
14. Count cancer cell number. Counting dead cells can be
$ |6 T* [9 _. c% _' N, ravoided by using Trypan blue staining. |
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发表于 2011-9-13 21:14
发表于 2011-9-15 14:05
