CGH Protocols （一）

Metaphasechromosomepreparation

Materials:RPMI1640mediumfetalcalfserum(FCS),20%Colcemid(e.g.BoehringerMannheimcellBIOLOGyreagents,Best.-Nr.295892)cellcutureflaskPhythemaglutinin,PHA-L(SEROmed,M5030)CO₂cellcultureincubator50mlNunc/Falcontubes15mlNunc/FalcontubesKCl(0.075M,0.055%?)Fixative(methanol/aceticacid3:1)glassmicroscopyslides

Amountsper5mlblood:40mlRPMI1640Medium10mlFCS(20%)5mlperipheralblood(anticoagulationbyheparin)1.5mlPHA1cellcultureflask,e.g.Falcon250mlprepareupto10flask(1flaskwillyieldabout50slides)

Steps:

1. Incubateculturefor72hoursinCO₂cellcultureincubator,mixflask1-2timesperday
2. AddColcemid(about45minbeforeharvesting)
3. Make2aliquotsandtransfercellinto50mlFalcontubes
4. Incubateincellcutureincubatoror37°Cwaterbathforadditional45min
5. Centrifugefor10minat1000rpm
6. Removesupernatante.g.withacellculturepipettoruntil5mlremain
7. Gentlyadd40mlKCl(0.075M,37°C),first5mldropbydrop(hypotonictreatment)
8. Incubatefor25minin37°Cwaterbath
9. Centrifuge10minat1000rpm
10. removesupernatant,leaveabout5ml,resUSPendpellet
11. Add2mlfixative,mixwell
12. Addfixativeuntil40ml,mixmeanwhile
13. Repeatsteps9-12untilthepelletiswhite(atleast4times)
14. Afterremovalandresuspensionofthepellet,transfercellin15mlFalcontube
15. Repeatsteps9-12,addjust10mlfixative
16. Removefixativeuntilabout2mlfinalvolume
17. Resuspendpelletandapplysuspensiononslides:

• Coolslidesto-20°C(e.g.putabout10slidesinacuvetteinthe-20°Cfreezerandkeepthecuvetteonicewhilepreparingthemetaphaseslides)

• takeoneslideandmoistenitbybreathingfromveryclose

• eitherdrop50-100µlofthesuspensionontheslideorapplythesamevolumetotheinclinedslide(thefastdraininganddryingofthefluidisusuallyanindicationforgoodspreADIng)

• letthesusensionbegintodry(thefluidfilmstartstoretract)
• puttheslidebrieflyin70%aceticacid

(theaceticacidstepisawashingstepinparticularforremowingthecytoplasm,inadditionitmayhelpforthespreadingofthechromosome;thecellmembranesattachtothesurfaceoftheglassslidesandaredisruptedbytheliquidflow;thetemperaturedifferencebetweenthecellandtheglassslidesmayhelpinthedisruptionofthecellmembranes.Iftheweatherconditionsarefavorablethe70%aceticacidwashingstepmaybeomitted;inourexperiencethebestmetaphasesspreadsoccurondryandsunnydays.

18.Airdrythechromosomeslide,checkforchromosomespreadingandcytoplasmdebrisinaphasecontrastlabmicroscope,adjustvolumeoffixativesothatthedensityofnuclei/metaphasesisappropriate

19.Iftheconditonsarefavorable,prepareabatchofmetaphasesspreads

20.Keepslideinaboxatroomtemperature(uptoabout1-2months);metaphasespreadsmaybekeptlongerat-80°Corin70%ethanolat4°C.

21.Keepfixativewithlymphcatesat-20°Cuntilthepreparationofnewslides.Addnewfixativeandwashcellbeforethepreparationofnewmetaphasespreads.