小鼠ES细胞核型分析

wqcyqy0811

请问不同的细胞做核型分析的步骤一样吗? 我要做小鼠胚胎干细胞的核型分析，看到一篇文献说是先把ES细胞种到载玻片上，再秋水仙素处理；但我看以前的贴子写到要离心重悬细胞，不知道是怎么回事，哪位朋友知道，能否帮帮忙，介绍一下具体的操作步骤。十分感谢！

iseeyou1210

ES cell 核型分析一般就是看染色体的数目是不是和标准的一致。做G带，也看不出什么更deep的change，而且小鼠的染色体还真的不好认。4 C6 k, l/ B& d5 f
good luck！

寒山拾得

G Banding
  M9 r5 U8 G/ _6 c10. Place suitably aged slides (see note below) in 2X SSC in a Coplin staining jar with a lid in a water bath at 60°C-65°C for 1.5 h. Then cool the slides to room temperature by running tap water over the closed jar. Transfer the slides to 0.85% (w/v) NaCl at room temperature for 5 min.
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  l* r6 T3 `6 ^$ }$ }+ WBefore G banding, slides should be “aged” for between 3 and 21 d by leaving them in a closed box at room temperature. Fresh slides give poor G-band resolution. Maximum G-band resolution is achieved at ~10 d after slide preparation. Beyond this time, resolution slowly decreases until, after several weeks in storage, the chromosomes either fail to band and stain uniformly or show “pseudobands” that are not significant to the standard idiogram.' D5 |$ d+ K: X. i
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11. Drain the slides by touching them onto filter paper. Place them on a flat surface and flood the chamber with 0.025% trypsin in 0.85% NaCl for 15-20 sec.
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The trypsin exposure time is critical: Underexposure preserves chromosome morphology but gives poorly differentiated bands, and overexposure distorts morphology and eliminates most of the bands. Optimum trypsin times are known to vary among laboratories. A test slide should be treated for the minimal suggested time of 15 sec to establish the best treatment time for the rest of the slides.
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; y2 N  r( k3 rIn the laboratory of E.P. Evans, the optimal trypsin exposure time has been established as between 15 and 20 sec for mouse chromosomes, irrespective of the source of the mitotic cells.
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12. Stop tryptic activity by placing the slides back into 0.85% NaCl. Then rinse slides in phosphate buffer (pH 6.8) and stain in fresh Giemsa stain in 5 mM phosphate buffer (pH 6.8). After 10 min in the stain, monitor wet slides under low-power, bright-field microscopy (160X) for staining intensity. Because, upon drying, wet slides gain contrast, care should be taken not to overstain the cells as this will reduce G-band differentiation. If necessary, repeat staining until adequate results are achieved and then quickly rinse slides in phosphate buffer (pH 6.8) and blow-dry with a current of cool air.
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! D) U' P, \" z( C' ~5 g! G13. Examine unmounted slides with a 100X oil-immersion lens.
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) F6 B. m; V2 `8 b$ u& s0 xThe majority of modern, readily available immersion oils that are declared “PCB-free” also have the unfortunate property of removing Giemsa stain after a few hours of exposure. Although direct viewing of slides under an oil immersion lens gives a higher optical resolution, it is wise to mount slides if they are to be kept.

寒山拾得

http://www.millipore.com/userguides/tech1/mcproto037
  k5 i* i+ c, BProtocol: Karyotyping ES Cells3 l+ k1 c) r/ H
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Catalogue Number:        mcproto037
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) r  |  k. O( J8 X! dYear:        2007       
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This method works best with actively growing culture of ES cells (i.e. 1-2 day culture).% p7 v; m( q  T1 Y+ @2 Z, K2 o* H
Method
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1.        The day before, passage a 70% confluent ES cell plate 1:2.
9 j- w+ H% {9 S% Y2.        On the morning of karyotyping, change the medium on the plate at least 3 hours before passage and collection.
) p& \! @* r% v9 C" e8 F$ |1 c" y3.        Trypsinize and collect ES cells into a conical tube. Centrifuge as usual and aspirate medium. Avoid allowing the pellet to dry out.
5 J" e" f* O( J$ \* |" ~$ G, q  W4.        Gently flick the tube to resuspend the cell pellet, and add 8mL of hypotonic KCl solution to the cells. Continue to gently flick the tube during the addition of KCl to avoid clumping.( L# W, _& ?' H3 }8 p' M( c
5.        Incubate the tube at 37°C for 10 minutes (this may vary for each type of cell line used).
' c* E- k5 z  `6 L6.        Add 2mL of freshly made fixative and mix by gentle inversion. {Fixative MeOH:Glacial Acetic acid 3:1 made fresh and stored at 4°C}.# X* h! ?8 i7 `
7.        Centrifuge cells at 1000 rpm for 5 minutes and aspirate supernatant.( a& |6 Q$ t6 e, F
8.        Using a pasteur pipette, carefully add 2mL of fixative solution dropwise, with gentle mixing to avoid clumping. Add an additional 6mL of fixative and mix by gentle inversion of the tube.  k8 _8 `8 Z8 b1 d
9.        Centrifuge cells at 1000 rpm for 5 minutes and aspirate supernatant.
  P1 r3 T' t2 F8 n% m' T2 ?10.        Repeat steps 8 & 9 three times.
& T- k+ _6 q$ O% Y6 |11.        Resuspend the pellet in 1mL of fixative (less or more according to pellet size).
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To make cell spreads, firstly humidify the surface of a dried cold slide by application of warm breath, whilst holding the slide at a 45° angle. Using a pasteur pipette, carefully drop (from a height of approx 0.5 metres) one drop of the suspended cells onto the top surface of the slide and allow to air dry.
! i, y- ~& c/ H$ bStaining4 D( p+ N* L( }1 c, A; y7 |) {2 i
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Stain slides with freshly made Leishmann’s stain for 8 minutes.* N' a* q7 N' V9 r' ]' U
Rinse in running water for 1 minute and air dry.
1 c! t4 a2 I2 a, fClear cells in 2x changes of xylene and mount coverslip using Depex.
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( }2 G2 L* Y- C. O" I9 b5 _% VColcemid is not used in this method, as the mitotic index of actively growing ES cells is generally high enough to get a good chromosome spread.
4 x2 M1 G) i' ZHigh quality slides need to be used. Slides should be soaked in 100% ethanol overnight and dried with lint-free tissue before use. As it is important to have slides “cold” before use, slides in ethanol bath can be stored in fridge or freezer until ready to make cell spreads.
2 i: `7 Y6 q3 A- hSome notes on KCl:- Most labs use 0.56 % KCl and some labs use 0.2% KCl + 0.2% Na citrate instead. This depends entirely on the cell types being analyzed. The time in KCl is crucial – too short and the chromosomes will be too tightly packed; too long and they will not remain in their appropriate group.

zhouwenping

拜托 大家都专业一点好不好，不同细胞的核型分析方法差别是很大的 ，不要拿着别人的步骤就给别人扣行不行，自己没有做过就不要在这里瞎说，会害惨很多人的，

hcluo

老化以后，就可以就行胰酶消化，Giemsa染色了。

hcluo

老化有助于G带显示，我们实验室一般将固定好的细胞滴在载玻片上，然后放在室温老化3-7天吧。

oucflora

干细胞的核型没有做过，不过正常体细胞核型分析时，不能老化，必须选择处于分裂增殖旺盛时的对数生长期来做，因为对数生长期的细胞增殖分裂比较旺盛，用秋水仙素处理，能获得较多的分裂相。如果你看的文献是针对于体细胞的，那么他这么说个人认为不合适。

wqcyqy0811

有的说还需要老化，请问在哪个步骤老化，老化是什么意思？怎么老化，必须老化吗？谢谢

wqcyqy0811

谢谢。十分感谢