Synchronizing Worm Cultures
来自 : 蚂蚁淘
SynchronizingWormCultures
Synchronizationisbasedonthefactnewlyhatchedlarvaewilllivebutnotdevelopifdeprivedofafoodsource.Anasynchronouspopulationofembryosisisolatedandthenallowedtohatchintheabsenceoffood.OncealltheembryoshavehatchedintoL1larvae,thenowsynchronouspopulationistransferredtomediumwithfoodandthepopulationoflarvaeresumetheirgrowthanddevelopment.Materials:
M9: | 22mMKH2PO442mMNa2HPO485.5mMNaCl1mMMgSO4 |
alkalinehypochloritesolution: | approx1%NaOCl(=140mM)250mMKOH |
sterileculturedishor24-wellplate | Procedure:- Collectgravidadults
- Harvestgravidadultsbywashingwormsoffwellpopulatedplateswith~1mlM9
- Collectin15mlcentrifugetubeorinEppendorftubesdependingonscale
- Pelletwormsbycentrifugation20-30secondsat150-200xg(30secondsat1200rpminclinicalcentifuge)
- Removesupernatant
- WashoncewithequalvolumeofM9
- Removesupernatant
- Isolateembryosfromadults
- Add1-5mlsofFRESHhypochloritesolutiontoworms(1mlforeppendorfor5mlsfor15-mlconicaltubes)
1ml: | 700ulsH2O | | 5mls: | 3.5mlsH2O | | 200uls5%NaOCl | | | 1.00mls5%NaOCl | | 100uls5MKOH | | | 0.5mls5MKOH | - Mixwellbyinversionandmonitorprogressunderthedissectingscope
- wormswilldisolveinthebleachsolutionleavingtheembryoswhicharemoreresistanttobleach
- Whenembryosarereleased(3-7minutes)collectbycentrifugation20-30secondsat150-200xg
- Removesupernatant
- Wash4XwithequalvolumeofM9
- ResUSPendwormsinsmallvolumeM9
- HatchoffL1s
- Transferembryostoculturedishorwellofa24-wellcultureplatewithsterileglassPipette
- Allowembryostohatchovernight(>12hours)at20degrees
- TiterL1populationbycounting"swimming"L1sin10ulspotsof10-folddilutions
- Transferdesiredamountofwormstomediumwithfoodsource
- Allowdevelopmenttodesiredstage
Notes:- H2OcanbeusedinplaceofM9
- sterilitydoesn"tmatteruntilafterthebleachingsteps
- agitationoftheembryosduringthehatch-offisunnecessary-theymightnotbeswimmingthenextdaybutarejustfine
- synchronizationisusuallyprettygoodthroughtheadultstage
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