Stock( i9 F& g& h1 j; j# v- U
concentration 6 v6 e% C$ Z, ]$ {+ o(mg/ml) 9 f. @& O1 N$ V- ]! ^ r% _- X1 ~Working9 i; L+ m0 l8 j/ ?. _( `- E8 d
concentration + b% w" C' K% k! ~. c% S& |/ k(mg/ml) $ K9 a; n. s# P# K! y1 |Volume0 q' m5 T; }. x- J F% I
(ml) ! Q6 Q# w6 O0 s7 C( o% w, \: J7 QCollagenase 200 0.4 0.1 9 d- j% A; d3 Q6 nDispase 200 0.5 0.125 1 F6 D& y5 h) KDNase I 100 0.2 0.15 e. }; y, B0 ?3 ~. W% \
Dulbecco’s phosphatebuffered 8 C" t- t) z) `* G5 v. Wsaline (DPBS)+ q2 ? ]. X" Q! p8 [
49.675 + b1 Z& n& b3 T: V, h% KCollagenase, dispase, DNase I stock . Store at −20°C. Stocks can be 0 U F3 p: W1 \# m7 tmade according to manufacturer’s instructions.! t, e5 Y% @) k7 M2 J8 I! ~2 M
Enzyme mixture solution: Make fresh as required, store . m6 ^( K5 U+ `2 kat 4°C.4 c( m6 L% H' a8 P1 L% {$ F6 m
2. Materials' q1 @: ^; g% f! [. l# W
2.1. Preparation of, [; t4 T8 n6 t) D. x( i
Cancer Samples . i3 ^2 ?( H$ t8 ~2.2. Dissociation of- |" f# ~$ R' ?' w H/ A' D$ w
Primary Cancer into 2 y" }3 `9 ]7 F/ _9 @Single Cells 9 }6 R& X' W: a8 a1 V D7 N6 o' x2.2.1. Equipment作者: amosummer 时间: 2011-9-15 14:07
3.2. Dissociation of Primary Cancer into Single Cells 6 J6 w0 W+ |" R8 I1. Transfer an extracted glioblastoma sample into 100-mm 2 4 I, Z; B# g9 {/ t" Tculture dish and then wash the sample several times with icecold # }0 I5 D3 G, U2 cDPBS. While washing the sample, remove blood cloths 3 {1 t' k1 Y( J+ }6 p, x% @) Band gross necroses. + N# ^3 {& ?( T" C/ u3 s2. Divide the sample into 1-cm 3 blocks using a scissor and; h7 L9 B D) d. ?3 i, ^, X( ], i
transfer them into 1.5-ml E-tubes. Samples should be in ; m/ a( r, v& f) O# Dice-cold DPBS during the procedure to prevent tissues from ( k, B& ?1 p: l% W7 v7 gbeing dried.! X& C3 f2 x# {3 ?, | }
3. Dissociate the blocks mechanically by chopping them many0 t: _' {% ~9 D; y
times with scissors as shown in Fig. 3a ., G( D# ]9 {9 y1 n
4. After primary mincing, collect the samples in a 15-ml conical( @. w" Q0 ^" W6 x: Q* p( x
tube or a 1.5-ml E-tube and triturate them further by sucking) r8 n( n% x6 ~6 S$ R. n
them in a Pasteur pipette multiple times as shown in Fig. 3 b 3 o5 y5 ?8 G3 b$ w( see Note 2 . for air bubbles). 7 S0 s8 B1 q; u$ ]5 c5. Wash the sample several times with ice-cold DPBS./ B1 W) u6 ~$ h c) f
6. Digest the sample using the enzyme mixture solution ( see% Y" G6 ]; q* J K
Note 3 . for alternative enzymes). The ratio of enzyme mixture- s0 T W) L, n0 L! m% ?4 O2 F
solution and mechanically dissociated sample volume is + ]; e' [$ D" y+ J4 l: a. F' M: \usually 1:1. The enzymatic digestion is persisted for 30–60 5 G" q j# t D' v2 j5 }% `min at 37°C. After the first 30 min of incubation, mechanical " {) K7 h0 S$ n4 g2 P4 V' |5 ?: Rmixing or mild trituration can be applied.1 b4 n* F6 c, D( Z
7. Wash the chemically digested sample several times with icecold; ?! T0 p Z o4 j& o1 J
DPBS.! _; z/ L5 l, C
8. Filter the mechanically and chemically dissociated sample 5 P7 ]4 Z9 A1 A2 }& Y E# ?through a cell strainer (40 μ m pore size) and collect single 6 ^4 E, m( D- d7 U" |cancer cells as shown in Fig. 4 . ( K& \' n r" N" G1 j7 j5 C9. Collected cancer cells can be contaminated by red blood, b3 N" b, q7 H( s3 b
cells (RBCs). To eliminate RBCs, carefully layer the cancer2 Z3 G8 L1 Z8 m4 | B5 a# p
cell suspension on 15 ml Ficoll-Paque PLUS (1.077 density) 6 o* x8 O; y- D8 L0 F6 f! win a 50-ml conical tube.$ O7 c/ s5 {* j m6 R
10. Centrifuge it at 400 × g (2,000 rpm) for 20 min at room 9 V3 c# ^2 \, G; btemperature without brake. 4 I4 e/ ^, D( l* P+ s8 ~11. Take the cancer cell layer (middle) between DPBS (upper)! F- S0 v) }+ v, [' m2 A4 L
and Ficoll-Paque PLUS (lower) using a Pasteur pipette. $ F9 u$ m# e5 O' H8 T1 v$ U3 p9 K& r12. Wash the cancer cells with ice-cold DPBS for several times.# \7 x5 G$ Y: ]6 {- z: h% \; x. L
13. If cell debris still remains, allow the cancer cell suspension to( [6 t, i Z( g$ Y2 a
stand for over 15 min at 4°C and remove 70% of upper portion + A/ j0 k8 B# z4 qof suspension containing unwanted cell debris. ' J9 v# K2 d+ I* L8 D5 o9 T14. Count cancer cell number. Counting dead cells can be+ | {8 b5 z% r! [- C3 f' v
avoided by using Trypan blue staining.作者: amosummer 时间: 2011-9-15 14:07
3.2. Dissociation of Primary Cancer into Single Cells5 c: Z) h8 n; D
1. Transfer an extracted glioblastoma sample into 100-mm 2 8 B7 z$ N" r! i) A( hculture dish and then wash the sample several times with icecold + E: [8 D: k8 f3 nDPBS. While washing the sample, remove blood cloths- c+ m# r5 H# ~3 ^
and gross necroses. : z' r6 ?6 Z; X0 X5 G O n# S7 L5 l2. Divide the sample into 1-cm 3 blocks using a scissor and ! Y6 k% R" n) r% E3 d! H& h' s/ Otransfer them into 1.5-ml E-tubes. Samples should be in * x6 x' ]8 S, ?0 v+ ^5 yice-cold DPBS during the procedure to prevent tissues from ! K U. U% ]/ \- ?7 M; Bbeing dried.( ^% ?( Y( r, T
3. Dissociate the blocks mechanically by chopping them many* R: `: e$ V- B% u1 T
times with scissors as shown in Fig. 3a . , _2 V/ a3 C8 x: J7 n4. After primary mincing, collect the samples in a 15-ml conical" z0 u% X6 m" E9 _8 z
tube or a 1.5-ml E-tube and triturate them further by sucking6 s3 d- a7 I- V1 R
them in a Pasteur pipette multiple times as shown in Fig. 3 b + y$ E( _7 ]+ k: b$ b( see Note 2 . for air bubbles). 0 o% T2 U' s, v$ C- |+ f5. Wash the sample several times with ice-cold DPBS.# Z) Y v4 W6 l9 n$ Y v9 f3 T
6. Digest the sample using the enzyme mixture solution ( see 3 y3 C% ~8 V; y& ANote 3 . for alternative enzymes). The ratio of enzyme mixture + h; f$ B4 T( d7 |- A: f0 Y, ^, qsolution and mechanically dissociated sample volume is) N7 M7 A7 Y1 d/ t3 D# ]- l7 i
usually 1:1. The enzymatic digestion is persisted for 30–60, T' Q# U/ h( V9 ?1 \/ H
min at 37°C. After the first 30 min of incubation, mechanical + r8 M9 @8 }6 ` f% a' z% omixing or mild trituration can be applied.0 ^. ]& N3 [8 D; l
7. Wash the chemically digested sample several times with icecold x! r' V7 j5 F/ D Y8 I8 d$ ~
DPBS. 6 a& C, J! s6 k7 E8. Filter the mechanically and chemically dissociated sample - }1 Z. e O& t" v$ H! |through a cell strainer (40 μ m pore size) and collect single 1 d& |( `. I% G! jcancer cells as shown in Fig. 4 . ! o. h: x2 @! h0 k1 w9. Collected cancer cells can be contaminated by red blood 4 x, C) V# `& O3 I9 l- gcells (RBCs). To eliminate RBCs, carefully layer the cancer + q; G h+ o) T7 a% Fcell suspension on 15 ml Ficoll-Paque PLUS (1.077 density) : a; C6 k. d& u% J1 s1 H1 kin a 50-ml conical tube. $ ?* f; N; s; {! M/ S; h10. Centrifuge it at 400 × g (2,000 rpm) for 20 min at room 4 k( K. Y8 B. R' X- xtemperature without brake. # K" ?" v$ R3 a. S4 H) T11. Take the cancer cell layer (middle) between DPBS (upper) 6 H, p- o$ A6 i% @1 c: {- L0 land Ficoll-Paque PLUS (lower) using a Pasteur pipette. w t) ~) ^: ?) j
12. Wash the cancer cells with ice-cold DPBS for several times.5 M: L- y3 K& A
13. If cell debris still remains, allow the cancer cell suspension to! b. u* B* V1 c9 B0 Z: E
stand for over 15 min at 4°C and remove 70% of upper portion 5 C: S( t4 s* z$ cof suspension containing unwanted cell debris.3 o! L2 T( L0 ^* d) c! x* C3 k
14. Count cancer cell number. Counting dead cells can be, a; c+ N4 D9 [; x; R
avoided by using Trypan blue staining.作者: pzypig 时间: 2011-9-24 21:58
