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发表于 2011-6-7 15:21 |只看该作者 |倒序浏览 |打印

之前有个人发了一个Nature protocol 上的染色体制片的英文帖子，其贴如下：Metaphase preparation from murine bone marrow2 F0 b0 @3 ?8 ]- a
Wei Guo, wguo@mednet.ucla.edu, University of California Los Angeles + Q; k4 }( G" t/ r
Hong Wu, wguo@mednet.ucla.edu, University of California Los Angeles 4 v$ K# D' g1 x8 @, d
Lab/Group: Wu Lab ) q% T$ m9 c0 L( M; |' w7 K# u8 i
Related Journal & Article Information
Journal: Nature ( i8 W2 [! B- ^4 \# g/ v
Article Title: Multi-genetic events collaboratively contribute to Pten-null leukaemia stem-cell formation  b5 q8 e# d1 b0 d, Y
Introduction9 B' _; E2 M+ O# @
Genomic instabilities including chromosomal translocations are frequently associated with genetic diseases and cancer, especially leukemia. Cytogenetic studies of these diseases requiring preparation of metaphase chromosomes are often key to revealing their chromosomal abnormalities. Treatment of in vitro disease cell cultures with cell cycle inhibitors, e.g. colcemid, have proved to be a very simple and effective metaphase preparation method for cytogenetic studies. Two good examples are metaphase preparation from cultured cells and some types of myeloid leukemia. However, this approach has some limitions and disadvantages: 1) it is difficult to grow many types of primary cell in vitro; 2) in vitro cultures tend to introduce additional artificial mutations to the genome. To overcome these issues, we have established an approach to prepare metaphase chromosomes directly from murine bone marrow. With this new approach, we were able to get 5-10% of metaphase chromosomes from primary bone marrow leukocytes and identify chromosomal translocations recurring in a Pten null murine model developing T cell leukemia (Figure 1,2 [1]). * w/ t9 c* t8 d! T
Materials
Reagents
1. 200 µg/mL colcemid (demecolcine, Sigma 27645, dissolved in 1x PBS, aliquotes are stored at -20°C) U: }, }$ N& u; h+ p# A
2. 0.06 M potassium chloride (hypotonic solution)$ ?0 b# d6 W  ^8 R- ~
3. Fixative solution (methanol [100%]:acetic acid [glacial]=3:1, fresh preparation required)$ B8 t' s  E8 ~. T1 o) }0 O
4. 20x Giemsa stain solution (Sigma GS500, freshly diluted)
5. 40 µm cell strainer (BD)
6. Pre-cleaned slides
Equipment2 Q' H  o6 F! D/ q, i& ^9 u( ?1 Q
1. 37°C water bath
2. 37°C incubator
3. slow rotator) @4 l' Q; v; {" S2 g9 Z9 o
3. gas or alcohol burner& J- T; ~9 |: D) L( w
4. phase-contrast microscope
Time Taken
It takes 2 hour to perform the experiment.
Metaphase preparation# y  b9 j/ Z: ?3 r) _
1|Grow adherent and or suspension cell cultures in their preferred media under their preferred growth conditions.. }) J. @9 [! @6 n; Y- G
In the case of adherent cell cultures, grow until they reach 60%–70% confluence (% confluence means the degree to which cells are making contact with each other). This determination is made using an inverted tissue-culture microscope.
2| Add colcemid (0.1 g ml–1, 10 μl for every 1 ml medium) to cell cultures using sterile micropipettes.2 b3 P; _+ [5 _2 i- [) z. e
▲ CRITICAL STEP For fast-growing cultures, reduce time of exposure to colcemid, and for slower-growing cultures, incubate for several hours with colcemid. Monitor cells throughout incubation period under the inverted tissue-culture microscope. Ideally, cultures with approximately 10–15 dividing cells per field of view (cells appear round and shiny) are ready for harvesting of metaphase chromosomes. For suspension and adherent cultures, one 75-cm2 flask should provide sufficient numbers of cells for preparation of metaphase spreads.# O" K6 @/ c1 V

收集细胞后4 M9 X$ @! d) E! x  t6 B
Procedure
1. Warm 0.06M potassium chloride in a 37°C water bath.
2. Inject 250µL of 200µg/mL colcemid into the peritoneum of mice and arrest cell cycle for 30 minutes.
3. Flush bone marrow cells with 30mL pre-warmed 0.06M potassium chloride into 50mL tube through 40µm cell strainer and rock sample tubes gently in 37°C incubator for 20 min.
4. Make fixative solution (methanol [100%]:glacial acetic acid=3:1) during hypotonic incubation., q, d% \' L! r% ^
5. Add 2mL Fixative into samples, mix gently. Centrifuge at 1500rpm for 7 min and discard supernatant.
6. Resuspend cells thoroughly with 0.5mL 0.06M potassium chloride.
7. Add few drops of fixative to cells and mix well. Keep adding and mixing fixative drops into cells until reaching 3mL. Add fixative up to 20mL in total, mix by inversion, and incubate at room temperature for 10 min. / N9 \, ^& e2 W; ^. O% |
8. Run the sample mixtures through 40µm cell strainer. Centrifuge at 1500rpm for 7 min and discard supernatant.( }0 ]6 Q" K1 }9 W" W0 |! A; a7 w) _
9. Resuspend pellets in 20 mL fixative and incubate at room temperature for 10 min. Centrifuge at 1500rpm for 7 min and discard supernatant.
10. Repeat step 9 once.
11. Resuspend cell pellets in 2mL fixative and store at -20°C for long-term storage.
12. To determine chromosome quality, use the flame method or other ways to spread and stain chromosomes on slides. Drop 30µL fixed cells onto pre-cleaned and wet slides tilted at a 45 degree angle. Put slides in burner flame for 2 seconds to allow chromosomes to spread out. Let slides dry." r% I1 E( `9 F* S' _2 C. x) Y
13. dilute 20x Giemsa stain with water to 1x Giemsa solution. Stain chromosome spread slides with 1x Giemsa solution for 5 minutes. Wash off Giemsa solution and let slides dry. Check chromosome quality under microscope.
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Troubleshooting
1Few chromosome spreads are available on slides. This is due to poor chromosome spreading by the flame method (Step 12). More practice runs should be conducted to improve spreading techniques. Alternative methods, such as dropping onto ice-cold wet slides from 30cm higher, are available for use, too.
Critical Steps
Step 7. The first few rounds of adding and mixing fixative drops are very critical for chromosome quality.7 w9 E5 H3 K6 g4 C$ K1 B+ b- X1 Y
Step 8. It is very important to remove cell debris through 40µm cell strainer beside filtering at Step 3. - e: E$ x$ e7 b* W5 b$ k
Step 12. Slides have to be rinsed and cleaned in distilled water before use. Otherwise, the quality of the Giemsa-stained slides will be compromised.
Step 13. Stain chromosome slides with 1x Giesma solution for 5 minutes at most. Excess staining time tends to result in overdarkened chromosomes# g. u" z# l% H5 J8 h  X
看着很费劲，我就又费劲的把它的大意翻译成了中文，翻过来后，还是觉得有些别扭，但还是希望对大家有帮助
中文翻译如下：
鼠骨髓分裂中期染色体制片6 l( K# p4 U/ U' \- T3 X
基因组的不稳定性（包括染色体易位）通常是与遗传疾病和癌症紧密联系的，特别是白血病。对于这些疾病细胞发生的研究，需要制备分裂中期的染色体，以显示其染色体的异常。在体外对疾病细胞用细胞周期抑制剂（如秋水仙素）处理，被证明是一种非常简单且有效的分裂中期染色体制片的方法。但是，这种方法有一些不足和缺陷：1.在体外各种类型的初生细胞生长很困难；2.体外培养易导致人工突变。为克服这些问题，我们建立了一种新的分裂中期染色体制片的方法。( P: b; z* D' [: \+ q* D& O2 E
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材料试剂' F0 |! W( b6 o6 Y' M0 q
1. 200 µg/mL colcemid (demecolcine, Sigma 27645, dissolved in 1x PBS, aliquotes are stored at -20°C, J- |  t. J7 @: W; L$ C( ^
2. 0.06 M Kcl (低渗溶液)
3. 固定剂 (甲醇 [100%]:乙酸 [glacial]=3:1, 必要的话可现制)2 j" ?: @) l& b7 |! X
4. 20x Giemsa stain solution (Sigma GS500, freshly diluted)* _3 b9 p; \; n* j- |: b
5. 40 µm cell strainer (BD)! B2 _& N- G( m" t4 ]
6. Pre-cleaned slides
仪器" A( m2 L( O2 G
1. 37°C 水浴% H* T% Q7 X' j
2. 37°C 保温箱
3. slow rotator" g3 F- @7 z# y- b% p
3. 酒精灯
4. phase-contrast microscope1 k- p' t% x% E  u' u1 W) ~8 W- j

整个实验过程大概需要2个小时6 q( [1 R% S( O" b4 h
1．在细胞适宜的介质和环境条件下培养细胞（贴壁或者悬浮的）。就贴壁的细胞而言，当生长达到60%–70% confluence时（%汇合表示细胞相互接触的程度），这通过nverted tissue-culture microscope确定。5 X( X& d/ S1 X" A
2．用灭过的微量吸液管加秋水仙素 (0.1 g ml–1, 10 μl for every 1 ml medium) 到培养的细胞中$ E  n' {; I9 G. S
▲ 关键步骤
For fast-growing cultures,减少秋水仙素处理的时间；for slower-growing cultures,用秋水仙素处理几小时也行。在整个孵育过程中用inverted tissue-culture microscope检测细胞。Ideally, cultures with approximately 10–15 dividing cells per field of view (cells appear round and shiny) are ready for harvesting of metaphase chromosomes. For suspension and adherent cultures, one 75-cm2 flask should provide sufficient numbers of cells for preparation of metaphase spreads.
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收集细胞后
步骤
1. 37°C水浴0.06M potassium chloride( U8 m/ b. {( A) ]# U% t$ E: R2 o& A
2. 于小鼠腹膜注射250µL of 200µg/mL秋水仙素，处理30min% i# j! q' w* e) E
3. 用30ml预热的0.06M KCL冲洗骨髓细胞至50ml的tube中（通过40µm细胞过滤器），然后在37°C保温箱中轻轻摇动样品for 20 min。
4. 在低渗孵育的时候，使固定剂溶解(methanol [100%]:glacial acetic acid=3:1)' b4 c: W) F" I0 g( B0 T, e
5. 加2ml固定剂于样品中，轻轻混匀。1500rpm离心7min，弃上清3 K+ J+ w$ Y. ?
6. 用0.5mL 0.06M potassium chloride重新彻底悬浮细胞' E. P5 [( s9 Z5 u+ k7 S# p- e  Z
7. 加几滴固定剂到细胞中，混匀。持续滴加固定剂并不断混匀，直至到达3ml。加固定剂到总体积为20ml，上下颠倒混匀，室温下孵育10min。& z7 F! ^( r' b" B( q
8. 样品混合液经40µm 细胞过滤器过滤。1500rpm离心7min，弃上清9 X; ]2 ^1 E( v, n8 n
9. 重新悬浮沉淀于20ml固定剂中，在室温下孵育10min，1500rpm离心7min，弃上清
10. 重复step 9 一次% ^1 d0 w& t0 |3 N. I  t$ S
11. 重新悬浮沉淀于2ml固定剂中，-20°C以长期保持。( S( Y4 X9 z5 X6 G" S/ K7 k& e
12. 为确定染色体质量，用flame method或其它方法在载玻片上spread and stain染色体。滴30µL混合细胞液到pre-cleaned and wet的在玻片上。将载玻片置于火焰中2秒，使染色体充分伸展开。使载玻片变干。
13. 用水稀释20x Giemsa stain 为 1x Giemsa solution。用1x Giemsa solution染色上述的载玻片5min。洗去Giemsa solution，使载玻片干燥，镜检。
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疑难解答$ ~/ g" k# F% t) p3 M  p
Few chromosome spreads are available on slides. This is due to poor chromosome spreading by the flame method (Step 12). More practice runs should be conducted to improve spreading techniques. Alternative methods, such as dropping onto ice-cold wet slides from 30cm higher, are available for use, too. , i$ q) j2 [+ [  Z; C" [
关键步骤
Step 7. The first few rounds of adding and mixing fixative drops are very critical for chromosome quality.
Step 8. It is very important to remove cell debris through 40µm cell strainer beside filtering at Step 3. / Z# Q! k! F& D% c
Step 12. Slides have to be rinsed and cleaned in distilled water before use. Otherwise, the quality of the Giemsa-stained slides will be compromised.
Step 13. Stain chromosome slides with 1x Giesma solution for 5 minutes at most. Excess staining time tends to result in overdarkened chromosomes4 |( J. w8 T& [( _3 p

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补充内容 (2011-6-7 15:27):
某些步骤或者过程可以根据实际优化，但总体过程基本一致