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- 积分
- 62
- 威望
- 62
- 包包
- 928
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http://www.millipore.com/userguides/tech1/mcproto037
0 w, {' a$ I z5 DProtocol: Karyotyping ES Cells
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; O* k: T( c5 XCatalogue Number: mcproto0377 j* }! O& l* {+ B* N: W. ]- f
( I! q0 `! `" k5 `3 rYear: 2007
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This method works best with actively growing culture of ES cells (i.e. 1-2 day culture).
( A1 G/ S) ^. RMethod
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$ H; E! r# G% `! V! I1. The day before, passage a 70% confluent ES cell plate 1:2." u& Z+ N, w$ r8 Z. C) ?) W5 b
2. On the morning of karyotyping, change the medium on the plate at least 3 hours before passage and collection.
- m# c# W0 O7 v+ M) {% P C+ \' b3. Trypsinize and collect ES cells into a conical tube. Centrifuge as usual and aspirate medium. Avoid allowing the pellet to dry out.) Z4 c7 l6 `. s
4. 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.
+ p- G: ?# |) B2 @5. Incubate the tube at 37°C for 10 minutes (this may vary for each type of cell line used).2 n9 B- R* a' I4 T
6. 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}.
+ `& S2 z5 ]) u) c6 \& n7. Centrifuge cells at 1000 rpm for 5 minutes and aspirate supernatant.
6 O) k# t X0 n& x/ z, M8. 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.; z; j" a+ P2 b
9. Centrifuge cells at 1000 rpm for 5 minutes and aspirate supernatant.
( P. ^0 ]- h) z' y+ y+ M10. Repeat steps 8 & 9 three times.
4 r/ k% F1 t2 p/ O3 E11. Resuspend the pellet in 1mL of fixative (less or more according to pellet size).- B( Z# q; H- p1 \& M5 V
4 B" W, n& Y0 i, v8 v5 XTo 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.
5 l2 U& p3 H" CStaining
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' d, H. Q+ h" i( w4 dStain slides with freshly made Leishmann’s stain for 8 minutes.
# |3 B6 E) g9 H8 N$ yRinse in running water for 1 minute and air dry.
( f/ z+ ^/ f. b; w ~' T6 RClear cells in 2x changes of xylene and mount coverslip using Depex.
/ P& `5 S e0 h5 @4 ?Notes
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7 q; Q1 L% h% l; C, h5 UColcemid 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.
0 ]/ {% w8 j& H3 IHigh 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.
% f; {% B7 C7 J. q- P" E1 L1 oSome 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. |
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发表于 2011-8-5 23:05
发表于 2011-8-8 19:21
