| Overview |  PrinterFriendlyVersion |
| Ex/Em(nm) | None/None |
| MW | N/A |
| CAS# | N/A |
| Solvent | N/A |
| Storage | F/D/L |
| Category | CellAnalysis CellCytotoxicity |
| Related | ApoptosisandCytotoxicity CellApoptosis BiochemicalAssays |
1. Culturecellstoadensityoptimalforapoptosisinductionaccordingtoyourspecificinductionprotocol,butnottoexceed2x106cells/mL(ornottoexceed3x105cells/100μL/wellina96-wellblackclear-bottomplate).Atthesametime,cultureanon-inducednegativecontrolcellpopulationatthesamedensityastheinducedpopulationforeverylabelingcondition. Hereareafewexamplesfor inducingapoptosisinsUSPensionculture:
1)TreatingJurkatcellswith2μg/mlcamptothecinfor3hours.
2)TreatingJurkatcellswith1μMstaurosporinefor3hours.
3)TreatingHL-60cellswith4μg/mlcamptothecinfor4hours.
4)TreatingHL-60cellswith1μMstaurosporinefor4hours.
Note:Eachcelllineshouldbeevaluatedonanindividualbasistodeterminetheoptimalcelldensityforapoptosisinduction.Foradherentcells,use2-3x104cells/welltostart.
2. Make150XTF3-DEVD-FMKDMSOstocksolutionbyadding200μLofDMSOtothevialofTF3-DEVD-FMK(ComponentA).
3. AddTF3-DEVD-FMKata1:150ratioand/orAnnexinV-iFluor488™(ComponentB)at1:100ratiointoeachwell,incubatethecellsina37°C,5%CO2incubatorfor1hour.
Note1:Thecellscanbeconcentratedupto~5X106cells/mLforTF3-DEVD-FMKlabeling.Theunused150XTF3-DEVD-FMKDMSOstocksolutionshouldbedividedassingleusealiquotandstoredat-20°C.
Note2:Foradherentcells,gentlyliftthecellswith0.5mMEDTAtokeepthecellsintact,andwashthecellsoncewithserum-containingmediapriortoincubationwithTF3-DEVD-FMK.
Note3:AnnexinVflowcytometricanalysisonadherentcellsisnotroutinelytestedsincespecificmembranedamagemayoccurduringcelldetachmentorharvesting.However,methodsforutilizingAnnexinVforflowcytometryonadherentcelltypeshavebeenpreviouslyreportedbyCasiola-Rosenetal.andvanEngelendetal(seeRefs1and2).
Note4:Theappropriateincubationtimedependsontheindividualcelltypeandcellconcentrationused.Optimizetheincubationtimeforeachexperiment.
4. Ifdesired,labelthecellswithaDNAstain(suchasHoechstforwholepopulationofthecellnucleusstain,orpropidiumiodidefordeadcellsifthecellslabelwithAnnexinV-iFluor488™only).
5. Spindownthecellsat~200gfor2minutes,andwashcellswithandwashcellswith1mL(or200μL/wellifusing96-wellplate)washbuffer(ComponentE)twice.Resuspendthecellsindesiredamountofwashingbuffer.
Note1:TF3-DEVD-FMKand AnnexinV-iFluor488™ arefluorescent,thusitisimportanttowashoutanyunboundreagenttoeliminatethebackground.
Note2:Fordetachedcells,theconcentrationofcellsshouldbeadjustedto2-5X105cells/100μLaliquotpermicrotiterplatewellforuseinstep6.
6. Monitorthefluorescenceintensitybyfluorescencemicroscopy,flowcytometer,orfluorescentmicroplatereaderatEx/Em=550/595nmforTF3-DEVD-FMK,490/525forAnnexinV-iFluor488™,350/461nmforHoechststain,and535/635forpropidiumiodide.
6.1 Forflowcytometry,monitorthefluorescenceintensityusingtheFL1channelforAnnexinV-iFluor488™,FL2channelforTF3-DEVD-FMK.Gateonthecellsofinterest,excludingdebris.
6.2 Forfluorescencemicroscopyandfluorescentmicroplatereader.Place100μLofthecellsuspensionsintoeachofwellsofa96-wellblackmicrotiterplate.
Note: Ifitisnecessarytoequilibratethecellconcentrations,adjustthesuspensionvolumefortheinducedcellstoapproximatethecelldensityofthenon-inducedpopulation. Thisadjustmentstepisoptionalifyourcelltreatmentdoesnotresultinadramaticlossinstimulatedcellpopulationnumbers.
6.3 ObservecellsunderafluorescencemicroscopeusingTRITCchannelforTF3-DEVD-FMK,and/orFITCchannelforAnnexinV-iFluor488™(TRITCchannelforpropidiumiodidestaining,DAPIchannelforHoechststaining).
6.4 MonitorthefluorescenceintensityusingEx/Em=490/525nm(cutoffat515nm)forTF3-DEVD-FMK,and/or550/595nm(cutoff575)forAnnexinV-iFluor488™ bottomreadmodeforafluorescentmicroplatereader.
| References&Citations |  CitationExplorer |
Anthocyanin-richblackcurrantextractinhibitsproliferationoftheMCF10AhealthyhumanbreastepithelialcelllinethroughinductionofG0/G1arrestandapoptosis
Authors:NaokiNanashima,KayoHorie,MitsuruChiba,ManabuNakano,HayatoMaeda,ToshiyaNakamura
Journal:MolecularMedicineReports(2017):6134--6141
ClusterinsignalsviaApoER2/VLDLRandinducesmeiosisofmalegermcells
Authors:MuhammadAssadRiaz,AngelikaStammler,MareikeBorgers,LutzKonrad
Journal:AmericanJournalofTranslationalResearch(2017):1266
DetectingApoptosis,Autophagy,andNecrosis
Authors:JackColeman,RuiLiu,KathyWang,ArunKumar
Journal:ApoptosisMethodsinToxicology(2016):77--92
AAT Bioquest常用产品报价单
| 产品名 | 货号 | 公司分类 | 商城分类 | 美金价 |
| AAT Bioquest/Fluorescein, disodium salt *CAS 518-47-8*/2/100 mg | 2 | iFluor Rapid Tests | 酸碱缓冲液 | 1088.00 | AAT Bioquest/14-3-3 ζ (Ab-58) Antibody/8B0001/50 ug | 8B0001 | Signaling Intermediate Ab | 重组抗体 | 2828.00 | AAT Bioquest/Opioid Receptor (Phospho-Ser375) Antibody/8A0022/50 ug | 8A0022 | Phospho-Specific Ab | 受体 | 2828.00 | AAT Bioquest/iFluor™ 350 goat anti-mouse IgG (H+L)/16440/200 ug | 16440 | Fluorescent Anti-IgGs | 荧光染料 | 1088.00 | AAT Bioquest/trFluor™ Eu goat anti-mouse IgG (H+L)/16518/100 ug | 16518 | Fluorescent Anti-IgGs | 抗小鼠 | 4278.00 | AAT Bioquest/RPE-streptavidin conjugate/16900/100 ug | 16900 | Streptavidin Conjugates | 其他生物染料 | 1378.00 | AAT Bioquest/GCNT3 Antibody/8C14708/50 ug | 8C14708 | Signaling Intermediate Ab | 功能性抗体 | 2828.00 | AAT Bioquest/Amplite™ Luciferase Reporter Gene Assay Kit *Bright Glow*/12518/1 plate | 12518 | Reporter Gene Enzymes | 其它检测试剂盒 | 2103.00 | AAT Bioquest/ReadiLink™ Rapid mFluor™ Violet 450 Antibody Labeling Kit *Microscale Optimized for Labeling 50 &mi | 1100 | mFluor™ Dyes and Kits | 标记试剂盒 | 2103.00 | AAT Bioquest/Amplite™ Colorimetric Urea Quantitation Kit *Blue Color*/10058/200 Tests | 10058 | Diagnostic Molecules | 定量试剂盒 | 2828.00 | AAT Bioquest/Screen Quest™ CHO-Gqi Chimera Cell line/38101/Each | 38101 | cAMP GPCR Assays | 常规细胞株 | 0.00 |
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