| Overview |  PrinterFriendlyVersion |
| Ex/Em(nm) | 484/520&630 |
| MW | N/A |
| CAS# | N/A |
| Solvent | DMSO |
| Storage | F/D/L |
| Category | MicroBIOLOGy FlowCytometry |
| Related | FluorescenceImaging |
| Spectrum | AdvancedSpectrumViewer |
Note1:Thawkitcomponentsatroomtemperaturebeforestartingyourexperiment.
Note2:TheKithasbeentestedatlogarithmicallygrowingculturesofthefollowingbacterialspecies:Micrococcusluteus,Staphylococcusaureus,S.warnerii,Bacilluscereus,Klebsiellapneumoniae,Escherichiacoli,andSalmonellacholeraesuis.
Note3:ManybacteriadonotshowaproportionalresponsetopartialmembranedepolarizationwithMycoStainIt™Green.Theresponseofeachbacterialsystemshouldbeinvestigatedandoptimized.OccasionallytheMycoStainIt™Greenconcentrationandstainingtimemustbeadjustedforoptimaldetectionofmembranepotential.Thefollowingistherecommendedprotocolforbacterialstaining.Theprotocolonlyprovidesaguideline,shouldbemodifiedaccordingtothespecificneeds.
Note4:Somecommonbuffercomponents,suchasTween20,sodiumazideandthimerosal,canaltermembranepotential,andshouldbeavoided.Besuretotestbufferadditivesfortheireffectonmembranepotentialduringoptimizationstudies.
1. Growbacteriainanyappropriatemedium.Bestresultsforhealthybacteriaareobtainedfromlog-phasecultures.Dilutethebacterialcultureto~106cellspermLinPBS(ComponentC)orequivalentsterilebuffer.Bacteriamaybediluteddirectlyfromtheculturemediumwithoutwashing.PreparesufficientsUSPensiontoprovide500µLpertest.
2. Aliquot500μlofthebacterialsuspensionintoaflowcytometrytubeforeachstainingexperimenttobeperformed.Preparetwoadditionaltubesforadepolarizedcontrolandanunstainedcontrol.
3. Add10μlof500μMCCCP(ComponentB)tothedepolarizedcontrolsampleandmix.
4. Add5μlof100XMycoStainIt™Green(ComponentA)toeachflowcytometrytubeandmix(donotaddstaintotheunstainedcontrolsample).Incubatesamplesatroomtemperaturefor30min.Stainedsamplescanbeanalyzedafter5min,butsignalintensitycontinuestoincreaseuntil~30min.
5. Stainedbacteriacanbeassayedinaflowcytometerequippedwithalaseremittingat488nm.Fluorescenceiscollectedinthegreen(fluoresceinfilter)andred(TexasRedfilter)channels. Theforwardscatter,sidescatter,andfluorescenceshouldbecollectedwithlogarithmicsignalamplification.
6. Instrumentadjustmentsareespeciallycriticalfordetectingrelativelysmallparticlessuchasbacteria.Usetheunstainedcontrolsampletolocatebacterialpopulationsintheforwardandsidescatterchannels.Usethesidescatterastheparameterforsettingtheacquisitiontrigger.
7. Applythedepolarizedcontrolsampleafteradjustingtheflowcytometerasdescribedabove.GateonbacteriausingforwardversussidescatterandadjustfluorescencephotomultipliertubevoltagessuchthatthegreenandredMFIvaluesareapproximatelyequal.Donotsetcompensation.
8. Whiletherelativeamountofredandgreenfluorescenceintensitywillvarywithcellsizeandaggregation,theratioofredtogreenfluorescenceintensitycanbeusedasasize-independentindicatorofmembranepotential.Thedatacanalsobeprocessedbygatingonbacteriausingforwardversussidescatter,andanalyzegatedpopulationswithadotplotofredversusgreenfluorescencereportingMFIvaluesaslinearvalues,notaschannels.
9. Onaratiometrichistogram,setMarkersaroundthepeaksofinterestandrecordthemeanratiovalues.Foradotplotofredversusgreenfluorescence,setregionsaroundthepopulationsofinterestandrecordredandgreenmeanfluorescenceintensity(MFI)valuesforeach.Toevaluatethedata,dividetheredpopulationMFIbythegreenpopulationMFI.
10. Intheflowcytometer,bacteriaareidentifiedsolelyonthebasisoftheirsizeandstainABIlity.Itisbesttoinspecteachsamplebyfluorescencemicroscopytoconfirmthattheparticlesdetectedareindeedbacteria.
| References&Citations |  PrinterFriendlyVersion |
1. MyintzuHlaing,M.;Wood,B.;McNaughton,D.;Ying,D.;Augustin,M.A.,RamanspectroscopicanalysisofLactobacillusrhamnosusGGinresponsetodehydrationrevealsDNAconformationchanges.JBiophotonics2017,10(4),589-597.
2. Shi,C.;Jia,Z.;Chen,Y.;Yang,M.;Liu,X.;Sun,Y.;Zheng,Z.;Zhang,X.;Song,K.;Cui,L.;Baloch,A.B.;Xia,X.,InactivationofCronobactersakazakiiinreconstitutedinfantformulabycombinationofthymoquinoneandmildheat.JApplMicrobiol2015,119(6),1700-6.
3. Booyens,J.;Thantsha,M.S.,Fouriertransforminfra-redspectroscopyandflowcytometricassessmentoftheantibacterialmechanismofactionofaqueousextractofgarlic(Alliumsativum)againstselectedprobioticBifidobacteriumstrains.BMCComplementAlternMed2014,14,289.
4. Zeidan-Chulia,F.;Keskin,M.;Kononen,E.;Uitto,V.J.;Soderling,E.;Moreira,J.C.;Gursoy,U.K.,AntibacterialandantigelatinolyticeffectsofSaturejahortensisL.essentialoilonepithelialcellsexposedtoFusobacteriumnucleatum.JMedFood2015,18(4),503-6.
5. DeLamo-Castellvi,S.;Toledo,R.;Frank,J.F.,ObservationofinjuredE.colipopulationresultingfromtheapplicationofhigh-pressurethrottlingtreatments.JFoodSci2013,78(4),M582-6.
6. Cai,P.;Huang,Q.;Walker,S.L.,DepositionandsurvivalofEscherichiacoliO157:H7onclaymineralsinaparallelplateflowsystem.EnvironSciTechnol2013,47(4),1896-903.
7. Nocker,A.;Fernandez,P.S.;Montijn,R.;Schuren,F.,Effectofairdryingonbacterialviability:Amultiparameterviabilityassessment.JMicrobiolMethods2012,90(2),86-95.
8. Lindback,T.;Rottenberg,M.E.;Roche,S.M.;Rorvik,L.M.,Theabilitytoenterintoanavirulentviablebutnon-culturable(VBNC)formiswidespreadamongListeriamonocytogenesisolatesfromsalmon,patientsandenvironment.VetRes2010,41(1),8.
9. Sawaya,K.;Kaneko,N.;Fukushi,K.;Yaguchi,J.,Behaviorsofphysiologicallyactivebacteriainwaterenvironmentandchlorinedisinfection.WaterSciTechnol2008,58(7),1343-8.
10. Alleron,L.;Merlet,N.;Lacombe,C.;Frere,J.,Long-termsurvivalofLegionellapneumophilaintheviablebutnonculturablestateaftermonochloraminetreatment.CurrMicrobiol2008,57(5),497-502.
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(1)、分子内含有发射荧光的基团，如羰基、氮氮双键、碳氮双键等。
(2)、分子内含有助色基团。助色基团使光谱红移并增大荧光效率，如伯胺基、仲胺基、羟基、醚键、酰胺基等。
(3)、分子内含有刚性平面结构的共轭π键。分子内共轭体系愈大平面性愈强其荧光强度愈高。一些能提高共轭度的因素能提高荧光效率，并使荧光波长向长波方向移动。 就是荧光染料的附着物，主要作用有帮助荧光染料展色、提高荧光染料与下游树酯的相溶性、保护荧光染料的性能。通常载体树脂是强极性树脂，分子中含有胺基、羟基、醚键、酰胺基等强极性基团，一方面有助色作用，增大荧光效率;另一方面与荧光染料有很好的相溶性，有助于染料的均匀分散。
荧光颜料常用的载体树脂有胺基树脂、苯代三聚氰胺一甲醛树脂、聚丙烯酸酯树脂、聚酰胺树酯、聚酯树脂、聚氨酯树脂等。 （1）、热塑性荧光颜料：线型
（2）、热固性荧光颜料： 体型
（3）、可溶解色精荧光颜料
（4）、水乳型荧光颜料 （1）、胺基树酯
（2）、聚酰胺树酯
（3）、聚酯树酯
（4）、丙烯酸乳液 （1）、塑胶类
低温型
中温型
高温型
（2）、涂料类
水性涂料
油性涂料
粉末涂料 （1）、含甲醛
（2）、不含甲醛
颜料是用着色的物质通常与被着色的物质混合在一起主要以无机化合物为主；染料是一种用来直接或经媒染剂作用而能附着在各种纤维和其他材料上的有色物质有的可以跟被染物质化合，多以有机化合物为主。 颜料不能上色,而染料能上色. 颜料和染料的区别 颜料是一种微细粉末状的有色物质,一般不溶于水、油和溶剂,但能均匀的分散在其中。颜料是色漆的次要成膜物质,在木材装饰过程中调制底漆、腻子以及木才着色,也经常使用颜料.
然后是碱性荧光染料，造纸厂用来增加纸的亮度。
直接和分散的荧光黄，印染厂用来增加棉质和化纤面料的艳度
希望同路人多多交流啊!
我想用共聚焦观察间期染色体，用涂染探针，有一个问题很困扰我，我想涂染完染色后用DAPI复染细胞核，但是目前哈尔滨的共聚焦都没有紫外激发光，不能激发DAPI，我怎么才能实现，用红色涂一条染色体，用绿色涂另一条，用DAPI蓝色染核，同时成像呢，所说的双光子显微镜可以么？我实在很迷惑，求求版主别再删我的帖子，尽管我现在只是一个索取者，还不能给大家提供有用的信息，但是相信有一天会为大家做贡献的，谢谢了
