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商品描述
Overview:
TheNeuroFreezekitcanbeusedtofreezeprimaryneuronsfromE18/19ratandP0/P1mousehippocampiandcortices.NeuronsfrozeninthismediamaintainhighlevelsofviABIlity(upto90%)uponthawing.Theseneuronsshowalltheexpectedcharacteristicsofprimaryneurons,includingappropriatemorphologyandexpressionofneuronalandsynapticMarkers.Theavailabilityofthismediawillenableinvestigatorstofreezeprimaryneuronsfromembryonicrats(E18)orP0/P1wildtypeorgeneticallyengineeredmice.
Data/Specifications:
Species: mouse,rat
How_To_Use:
FreezingofPrimaryNeurons:
- Obtaindissociatedprimaryneuronsfromrat/mousehippocampus/cortexasdescribed(Beaudoinetal).Re-sUSPenddissociatedcellsinminimalvolumeofplatingmedia.
- EstimatecellviabilityonaHemocytometer.
- Donotexceedamaximumof4millioncells/vialforfreezing.Itisrecommendedtofreeze1to4millioncellspervial.
- Re-suspendcellstobefrozenin900μlofReagentA.
- Add10%(100μl for1mL)ofReagentBtothevial.
- Add1%(10μlfor1mL)ofReagentCtothevial.
- Gentlymixandplacethevialinisopropanol bathin-80°Cfreezer.
- Nextday,placevial(s)in liquidnitrogen.
THAWINGPROTOCOL
- Followyourlaboratory’sprotocolforcoatingsubstrate.
- Addpre‐maintenancemedia(ReagentD)todishes/platesandincubatein37°Cincubatorforaminimumof2hours(recommended4hours).
- Bringavialofcellsfromliquidnitrogenandquicklythawin37°Cwaterbath(usuallytakesabout90seconds)–itiscriticalthatthawingisaccomplishedasfastaspossIBLe.
- Estimatecellviability.
- Seedcellsontothesubstrate(SeePlatingDensityChartbelow)
- Aftercellshaveattachedtothesubstrate(3-4hours),replacepre-maintenancemediawithyourlab’sneuronmaintenancemedia(B-27containingmaintenancemediaisrecommended).
- MaintainneuronsinB27containingmaintenancemediaat37°C.
