BD公司流式细胞仪LSR 说明书

รԫີΕΕBD LSR IIߓอิګຝ։
รԫີ
BD LSR IIੌڤาઽᏚ
BD LSR IIਢ׈੺Ղรԫ؀ױ಻ໂ؄֭ࡐࡳீᄷऱΕ௛ܐڤሼ୴ऱோՂীੌڤาઽᏚΔ،౨ٵழ։࣫ڍመ10ۥᘛ٠೶ᑇΖႃᨋ੒࣐شΕUVሼ୴Εᑇڗ֏ሽ՗ߓอ࣍ԫߪऱBD LSR IIΔױየߩޓᐖऑऱڍۥੌڤ։࣫Δࠌ൞່Օૻ৫چႃխતԺ࣍ઝᖂઔߒΔۖլਢᓤᠧऱຌ࿏᧯ᖙ܂Ζ
Ꮪᕴऱ๻ૠு֨ਢࠡᄅڤᑇۯ֏ᑇᖕ๠෻ߓอΔࠌBD LSR IIګ੡ഄԫటإऱᑇۯ֏ੌڤาઽᏚΖBD LSR IIऱᑇۯ֏ߓอױኔ෼ޢઞᤪ๠෻ԫեᆄڻऱ೏ຒ։࣫ΔٵழᏚᕴ଀ᄅ֏ऱ౧ᓢ๠෻ݾ๬Δຏመ։࣫ॾᇆ౧ᓢऱ૿ᗨΕᐈ৫Δ೴։ኔ᧯ิ៣ᑑءխऱ⊺ຑาઽᆢ᧯Δ່Օ࿓৫چ྇֟೗ၺࢤΔਢኔ᧯ิ៣DNA։࣫ऱึᄕᇞެհሐΙኙࠟጟᘛ٠ॾᇆֺ෷ऱૠጩٍױش࣍ၩᠦ՗ੌ೯ऱᛀྒྷΖ
BD LSR IIऱ٠ᖄ᧜ፂߓอലޢԫ֭ሼ୴٠ޔતᒔΕ᡹ࡳچႪፋڇ٠ޔݮګ④ᢴՂΔ٦ຏመ④ᢴΔലሼ୴٠ޔދ୴۟ፋྡྷຘᢴՂΖॾᇆگႃߓอ๯๻ૠګԶߡݮඈ٨Δຍጟ๻ૠױ່Օ࿓৫چൕޢԫଡሼ୴ᕴխᛀྒྷ٠ॾᇆΖޢԫଡPMTհছऱ൅ຏៀ٠ׂΔւ๺ش֪ኙࢬᏁگႃऱ٠ᢜᒤ໮೚તาᓳᆏΖط࣍֘୴ֺຘ୴ऱய෷ޓ೏Δࢬאຍጟ๻ૠ༼೏ԱᇠᏚᕴڇڍۥ։࣫ழऱᛀྒྷࢤ౨Ζ
ᨋ੒ࢤΚΚBD LSR IIױ୲౏1۟4 ௅ሼ୴Ζࢬڶሼ୴ᕴຟਢ௛ܐ׊൅ࡐࡳீᄷΔࠀ಻ໂ೯ ᨋ੒ࢤ
ኪᒤ໮ઌᔞΕᚌ။פ౨ऱPMTΖ
ሼ୴ΚΚଈڻ಻ᆜ࣍ோՂীੌڤาઽᏚΔࡐࡳீᄷΔ୲࣐ࠌشΖױൄ๵ش࣍ Indo-1 ྒྷࡳΕ UVሼ୴
Ca+ ຏၦΕDAPI ࢨ Hoechst ։࣫੒าઽDNAΖ
ڍۥ։࣫ΚΚᘛ٠਩ᕪᙇᖗᒤ໮ᏺףΖೈԱᑑᄷ 488nm ᖿ࿇ऱᘛ٠਩ᕪհ؆Δᝫڶ UV ᖿ ڍۥ։࣫
࿇ऱᘛ٠਩ᕪڕ DAPIΕHoechst 33342ΕIndo-1ΕGFPΙHeNe ᖿ࿇ऱᘛ٠਩ᕪڕ APC ࿛Ζߓอഗء࿨ዌΚΚ
1.1 BD LSR IIߓอഗء࿨ዌ
BD LSR IIาઽ։࣫Ꮪ׌᧯
FACSDivaሽᆰՠ܂ీ
BD
ੌڤาઽᏚץਔ෈ੌߓอΕ٠ᖂߓอΕࡉሽ՗ߓอΖৱྒྷᑌءխऱาઽᆖ෈ੌߓอႚಬࠉݧچຏመาઽᏚխሼ୴ᅃ୴ऱ೴഑Δาઽ࠹ሼ୴ऱᖿ࿇ขسॾᇆΔ๯ॾᇆ൷گᕴ൷࠹ࠀ࣋ՕΔຍࠄ࣋ՕԱऱॾᇆᆖሽᆰ։࣫๠෻Δࠀאቹ।ऱݮڤऴᨠچ᧩قנࠐΖੌڤาઽᏚขسࠀ։࣫ऱॾᇆ׌૞ڶ٠ཋ୴ॾᇆࡉᘛ٠ॾᇆΖ٠ཋ୴ॾᇆऱൎஇױא֘ਠาઽऱՕ՛Εݮኪ֗ઽᑠឍศ֏ऱ࿓৫࿛Ζࠉᘛ٠ైऱլٵΔشլٵं९ऱ٠ᖿ࿇Δ࿇୴נլٵं९ऱᘛ٠Δױ᧩قլٵऱᠱۥΔڇլٵऱኔ᧭᧯ߓխΔຍࠄᘛ٠ॾᇆ༉ױא֘ਠլٵऱาઽسढᖂ௽ࢤΖ
าઽᏚ׌᧯
LSR IIาઽᏚ
׌᧯ΚΚ
׌᧯
إ૿قრቹ
BD LSR II׌ᖲิګ։
1.2 Fluidics ෈ੌߓอ
෈ੌߓอץܶSample injection tube ᑌ঴Ղᑌಾ(SIT)Εflow cell ੌ೯৛Εࠟଡᚏ෈࿟Ε֗ԫߓ٨෈᧯ᙁಬጥᒵΖ෈ੌߓอऱ׌૞פشΔਢലาઽࠉݧಬࠩྒྷၦ೴࠹ᛀΖ
1.2.1 ੌ೯৛Flow Cell
LSR II าઽᏚආشയຨڤ෈ੌߓอΖڼԫߓอऱ෈ੌᚘԺፖᙫ෈ੌੌຒઃ੡଺ᐗቃ๻ΔֹᏁԳ੡ᓳᖞΔਚۖᖙ܂១໢ΖףՂᏚᕴփհሼ୴٠ሁٍ੡଺ᐗቃ٣ᓳ๻Δڂڼᔞش࣍ᜯݩᛀ᧭ፖഗ៕ઝઔΖ
܃ലڜᇘᑌ঴ጥڇSIT ՂΔ෈ੌߓอᇙԫଡᚥ௥ᄎףᚘᙫੌᙓ࿟Δ༼ࠎᙫੌࠩੌ೯৛խΖڇຍٵழΔᑌ঴ᇢጥՈ๯ך௛ףᚘΔۖᑌ঴ঁᄎႉထSIT ๯ಬၞੌ೯৛Ζԫ؟ᑌ঴ၞԵੌ೯৛Δาઽࢨศ՗ലݮګԫሁ᜕ၷٻሼ୴٠ޔฝ೯Ζࠐ۞ຍࠄศ՗ऱཋ୴٠ࡉ࿇נऱٺۥᘛ٠ല༼ࠎᣂ࣍،ଚऱ֡՚ΕݮणΕឍศࢤΕࡉس֏س෻௽ࢤऱಛஒΖڇੌ೯ᛀྒྷ৛խΔຘᢴലሼ୴٠ޔፋྡྷڇᑌ঴ੌၗ֨ۯᆜΖሼ୴٠ޔፋྡྷڇᑌ঴ੌՂขس່ࠋऱ٠ॾᇆΖط࣍٠ᖂຏሁࡉᑌ঴ੌၗ֨ऱۯᆜࡐࡳΔࢬאڼጟ່ࠋ֏णኪԫऴঅ਍᡹ࡳΖ
ੌ೯৛ੌ೯৛ଳ૿ቹଳ૿ቹଳ૿ቹΚΚ
ੌ೯৛փךየᙫ෈Δᑌ঴ᇢጥխऱาઽ๯ԫ೏࣍ᖞଡ෈ੌߓอऱᚘԺංԵੌ೯৛խΔݮګԫპาֽሁΔาઽױ࣍ࠡփٻՂཾ೯Ζ៶طੌ᧯೯Ժፋྡྷ଺෻ΰhydrodynamic focusing αΔױࠌᇠֽሁࡐࡳ࣍ੌ೯৛խ؇Δࡌ໮հᙫੌ෈ݮګԫץ๯ֽሁհᙫᓂΔࠌาઽ౨א໢ԫาઽհݮኪΔດԫੌᆖ່ాऱֽሁࠩሒྒྷၦ೴Δۖፖሼ୴٠ٌᄎΖ
෈ੌߓอقრቹ
1. ሽᄭၲᣂΚሽᄭڇᏚᕴ׳ছՀֱΖ
2. Ꮪᕴ૿ࣨΚ
ڇᏚᕴছֱ૿ࣨխ׽ڶ3ଡੌຒ൳ࠫ᝶ΰLO/MED/HIα֗3ଡפ౨൳ࠫ᝶Ζ
ੌຒ൳ࠫΚ
LOΚᑌ঴ੌຒΚ12 μl /min
MEDΚᑌ঴ੌຒΚ35 μl /min
HIΚᑌ঴ੌຒΚ60 μl /min
ੌຒპᓳඝၨΚֱؐհSample Fine Adjust ඝၨױპᓳ෼ڶᑌ঴ੌຒΔൕ50%-200%Ζ
LOΚᑌ঴ੌຒΚ6 - 24 μl/min
MEDΚᑌ঴ੌຒΚ17.5 - 70 μl/min
HIΚᑌ঴ੌຒΚ30 - 120 μl/min
෈ੌߓอפ౨൳ࠫΚ
• RUNΚጸۥழ।قᑌ঴ၲࡨᙁࣹΖΰᖪદۥழ।قᏚᕴլإൄΔᓮᛀ਷ਢܡዥ௛α
• STANDBYΚྤᑌ঴ࢨᄊᖲழհإൄۯᆜΔڼழሼ୴פ෷ᄎ۞೯૾܅א࢏९ሼ୴ኂࡎΖ
• PRIMEΚPrimeᖙ܂౨ඈೈੌ೯৛փऱ௛ऐΔشֲ࣍ൄൄߠംᠲऱᇞެΖଈ٣ᙫ෈ೖַੌ೯ΔᚘԺ֘ٻലᙫ෈ඈנੌ೯৛ੌԵᐒ෈දΖڇቃ๻ऱԫࡳழ
ၴ৵Δߓอאԫࡳऱຒ৫۞೯ലੌ೯৛ךየᙫ෈ॴַ௛ऐऱݮګΖՂ૪࿓ڤ
ݙګ৵ΔᏚᕴᄎၞԵStandbyᑓڤΖࠌشழᖲڕΚᄅᇘᖲၲᖲΕޓངPBSΕ
փຝጥሁڶ௛ऐΕ৺ੑᏚᕴࢨ৺ੑၞᑌಾ࿛Ζ
3. ᚏ෈ᙓ࿟Κ
ᙫ෈࿟Κᙫ෈ᙓ࿟୲ᗨપ8֒Δאጸۥጥᒵፖ׌ᖲઌຑΖᇘየᙫ෈࿟ΔᏚᕴױאሎ۩Օપ8՛ழΖᙫ෈࿟።Ղڶ८᥆ᛩڬΔঅဳᙫ෈࿟യຨΖᙫੌ෈࿟ڶۭᜤᙫ෈መៀᕴSheath FilterΔ0.22umመៀᕴΔװೈᙫ෈խऱᠧᔆΔঅᢞၞԵੌ೯৛ऱᙫ෈ਢ೓෣ऱΖᓮࣹრ௛ሁ྇ᚘᎺհۯᆜΖຍԫীऱ๻ૠ޲ڶᇘ෈૿ႚტᕴΔࢬאྤऄൕᏚᕴຌ᧯Ղቃवᙫ෈ܛലشݙΖ׽౨ش֫ᄀ೯ᙫ෈࿟ΔᖇૹၦტࠐܒឰΖ
ᐒ෈࿟Κ୲ᗨપ16֒ΔႿᓄޗᔆΔאᖪۥጥᒵፖ׌ᖲઌຑΖ࿟Ղᇘڶ෈૿ტᚨᕴΔᐒ෈ךየழᄎ࿇ᜢᤞܫΖش֪ױאؾီ۷ጩᐒ෈࿟ऱ۾የၦΔࠐංጩᙫ෈ऱໍਲၦΖ
4. Ղᑌ঴೴Κ
Ղᑌ঴೴ਢᑌءጥऱՂᑌۯᆜΖ،ץਔԿଡຝ։Δԫଡਢၞᑌಾ SITΔᝫڶ֭ᐶਮTube Support ArmΕࡉ෈ዠژఎߓอDroplet Containment Module(DCM)Ζ
•ၞᑌಾΚਢԫ௅լ᤬ᙓጥΔലาઽൕᑌءಾխܮԵੌ೯৛Ζၞᑌጥ؆ڶԫ୚ጥΔਢ෈ዠঅఎߓอऱԫຝ։Ζ
•֭ᐶਮΚ૤ຂඔ೯෈ዠژఎߓอΖ֭ᐶਮۯ࣍ؐೡࢨ׳ೡழඔ೯෈ዠژఎߓอΙ֭ᐶਮڇՂᑌणኪ੡ᣂຨΖ
•෈ዠژఎߓอΚߓอط֭ᐶਮΕట़ᚥ௥ࡉ؆୚ጥิګΖᅝ֭ᐶਮۯ࣍ؐೡࢨ׳ೡۯᆜழΔట़ᚥ௥༉ᄎඔ೯Δല෈᧯ൕ؆ጥܮԵᐒ෈࿟փΖՂᑌழΔႊ߰ຒല֭ᐶਮۯ࣍խۯΔאᝩ܍መڍᑌ঴๯ࢼܮࠩᐒ෈࿟փΔᅝ֭ᐶਮۯ࣍խۯΔట़ᚥ௥ೖַՠ܂Ζޓངᑌ঴ழΔᨃᏚᕴঅ਍RUNऱᑓڤΔࠌ൓ၞᑌಾױאܛࠥ֘ޱՂᑌΖ
ඔ೯DCM ᣂຨDCM
5. HTS (High Throughput Sampler) Ղᑌߓอ
BD LSR IIױᙇ಻HTSՂᑌߓอΔگႃ96ࢨ384პ֞ᒌհᛀ᧯Ζ
ڇᇘ಻ڶHTSհᏚᕴՂΔᄎڶԫگ࠷ᑓڤ֊ང᝶(Acquisition Mode Switch)ࠎࠌشृ֊ང໢ጥࢨHTSՂᑌᑓڤΖ!
•໢ጥᑓڤΚڇڼᑓڤՀDCMפ౨إൄΖᅝ֭ᐶਮۯ࣍ؐೡࢨ׳ೡۯᆜழΔట़ᚥ௥ᄎඔ೯Δല෈᧯ൕ؆ጥܮԵᐒ෈࿟փΖ
•პ֞ᒌՂᑌᑓڤΚڇڼᑓڤՀDCMפ౨ᣂຨΖ֊ང۟ڼᑓڤழΔၞᑌಾᄎڂDCMפ౨ᣂຨۖขسዠֽ෼ွΔዠՀհᙫ෈ᄎ๯Հֱհܮگቾ(adsorbent pad)ܮگΔڼ੡إൄ෼ွΔլᏁᖜ֨ΖޓངHTSറشհၞᑌಾঅᥨ୚ࠀലᛀ᧯ຑ൷ᕴ(sample coupler)ڜᇘݙฅ৵Δዠֽ෼ွܛᄎ௣؈Ζ
HTSዌທقრቹ:
1.3 Optics ٠ᖂߓอ
BD LSR II ߚᵤ۔Ոܝᅌிవᰃϵ▔Հܝᅌிవ੠ᬊ▊ܝᅌிవ᠔ఈ៤DŽ▔Հܝᅌிవ⚎⌕ࢩՈ௴ྡྷᐊ՚ܝ࿁˗ᬊ▊ܝᅌிవࠛᬊ▊ᕲ௴ྡྷ៪ୖᄤՀߎՈᙦܝֵᔣ៪້ᬷᇘܝDŽ٠ᖂߓอ٠ᖂߓอଳ૿ቹଳ૿ቹଳ૿ቹΚΚ
1.3.1 Excitation Optics ᖿ࿇٠ᖂߓอ
٠ॾᇆᖿ࿇ߓอطሼ୴ᕴΕ٠ᖄ᧜ፂΕݮګ٠ޔऱᆆᢴࡉԫଡ೏ຘ٠ࢤፋྡྷຘᢴิګΖ٠ᖄ᧜ፂߓอലሼ୴٠ޔતᒔΕ᡹ࡳچႪፋڇ٠ޔݮګᆆᢴՂΔ٦ຏመᆆᢴΔലሼ୴٠ޔދ୴۟ፋྡྷຘᢴՂΔߠՀቹࢬقΖ
LSR IIሼ୴Κ
BD LSR IIᏚᕴࠌش܅פ෷Ε௛ܐڤࡐኪሼ୴ᕴΔຍᑌԫࠐΔᇠᏚᕴ༉լᏁ૞௽௘ሽᄭࡉܐথ๻ໂԱΖ
ሼ୴ं९
ं९ΰΰnmα່՛פ෷
ΰmWα
ൄشᘛ٠਩ற
Coherent R Sapphire TM ࡐኪሼ୴488ΰ៴٠α20 FITC, PE, PE-Texas Red, PerCP,
PerCP-Cy5.5, PE-Cy7,PI
JDS Uniphase TM HeNe 633ΰદ٠α 17 APC,
APC-Cy7
Coherent Vioflame
ࡐኪ
405 (࿫٠) 25 Cascade Blue, Alexa 430 Kimmno HeCd 325 (࿫؆٠) 8 Indo-1, DAPI, Hoechst Lightwave X-Cyte
ࡐኪ
355 (࿫؆٠) 20 Indo-1, DAPI, Hoechst
1.3.2 Collection Optics گႃ٠ᖂߓอ
ፋྡྷຘᢴߓอঞ֧ᖄሼ୴٠ၞԵੌ೯৛ு֨ऱᑌ঴೯ᒵՂΖא 6-2 ๵௑๻ૠLSR_II੡ࠏΔگႃ٠ᖂץਔࠟଡᛀྒྷᕴೄ٨ΔطڕቹխࢬقऱԫଡԶߡݮࡉԫଡԿߡݮඈ٨ऱ٠ሽጥิګΖ
Զߡݮᛀྒྷᕴೄ٨ץܶԮଡ٠ሽጥΔ،૤ຂᛀྒྷࠐ۞៴٠ሼ୴ऱॾᇆΖࠡխڶԫଡ٠ሽጥΔ،૤ຂᛀྒྷ90৫ೡֱཋ୴٠ॾᇆΖԿߡݮᛀྒྷᕴೄ٨ץܶࠟଡ٠ሽጥΔ،૤ຂᛀྒྷࠐ۞દ٠ሼ୴ऱॾᇆΖ
٠ᖂಛᇆ൷گᕴቹق(ቹ6-2 ๵௑)Κ
ॾᇆگႃߓอ๯๻ૠګԶߡݮࡉԿߡݮऱඈ٨Δຍጟ๻ૠױڶயچൕޢԫଡሼ୴ᕴխᛀྒྷ٠ॾᇆΖࠠ᧯ڕՀΚຏመԫิ९ຏԲ։ᢴLongpass DMΔ٣ലᘛ٠ॾᇆխं९່९ऱႚᙁࠩรԫଡ٠ሽጥΰPMTαՂΔ֘୴ڃࠐऱለ࿍ं९ऱ٠ॾᇆঞٻՀԫଡPMTֱٻ࿇୴ΖޢԫଡPMTհছऱ൅ຏៀ٠ׂbandpassւ๺ኙࢬᏁگႃऱ٠ᢜं९೚તาᓳᆏΖط࣍֘୴ֺຘ୴ऱய෷ޓ೏Δࢬאຍጟ๻ૠ༼೏ԱᇠᏚᕴڇڍۥ։࣫ழऱᛀྒྷࢤ౨ΖڕቹقΔBlue Aΰ750~810 nmαΕBlue Bΰ675~715nmαΕBlue Cΰ650~670nmαΕBlue Dΰ600~620nmαΕBlue E ΰ562~588nmαΕBlue Fΰ515~545nmαΕBlue Gΰ488nmαΖ
OctagonΚ
TrigonΚ
ڕቹقΔRed Aΰ770~830 nmαΕBlue Bΰ650~670nmα
ೈ٠ሽጥᛀྒྷᕴհ؆Δፖሼ୴ؓ۩ֱٻڶ࣋ᆜ
ԫ೏ඕ৫٠ሽԲᄕ᧯photodiodeΔشאگႃൎՕऱ
ছֱཋ୴٠ॾᇆΖຍଡԲᄕ᧯ছֱڶଡᔟ٠ཧΔױ
א߻ַإٻऴ୴ऱሼ୴٠ၞԵΔ׽൷گ៥୴٠Ζ
ៀׂ
ੌڤาઽ๬խࢬشៀׂڶৰڍጟΔڶ൅ຏៀׂΕ९ंຏៀׂΕ࿍ंຏៀׂΕ९ंຏᠨۥࢤ֘୴ׂΕխࢤៀׂΖ൅ຏៀׂऱᚨشঞإړઌ֘Δ،׽ւ๺ਬԫ௽ࡳं९ऱ٠ᒵຏመΔࠏڕԫ530 nm൅ຏៀׂ׽ւ๺FITC࿇୴ऱ530 nmጸۥᘛ٠ຏመΔۖࠡהं९ऱᘛ٠٤ຝ๯ॴឰΖ९ंຏៀׂ׽ւ๺ਬԫं९אՂऱ٠ᒵຏመۖലڼं९אՀऱ٠ᒵៀൾΔڕ650nm ९ंຏៀׂ׽ւ๺PerCP࿇୴ऱદۥᘛ٠ຏመΔۖ488nmऱᖿ࿇٠֗FITC࿇୴ऱ525nmጸ٠٤ຝ๯ॴΖ९ंຏԲۥࢤ֘୴ᢴ׽ւ๺ਬԫं९אՂऱ٠ᒵຏመۖലڼं९אՀऱ׼ԫ௽ࡳं९ऱ٠ᒵ֘୴Ζࠏڕᨃ620nmאՂऱદ٠ຏመۖല 525nmጸ٠֘୴Δࠡהऱ٠ᒵঞطᢴׂܮگΖխࢤៀׂྤ᙮෷௽ࢤΔ،ኙٺं९ऱ٠ᒵ݁֌ಐ྇Δൄش٠ᒵመൎᏁ૞݁֌ಐ྇ऱچֱΖ
ሼ୴٠ᄭ֗ኙᚨ൷گᕴᘛ٠ं९। ( 4-2-2-2 ๵௑)Κ
1.4 Electronics ሽ՗ߓอ
ሽ՗ߓอ૤ຂނ٠՗ॾᇆ᠏֏ګሽ՗ॾᇆΔࠀലհᑇۯ֏੡৵ᥛሽᆰ։࣫܂ᄷໂΖڇԲᄕ᧯ࡉPMTs٠ሽጥࢬขسऱॾᇆΔᄎፖהଚೠྒྷࠩ٠՗ऱᑇၦګإֺΖาઽᏚऱሽ՗ߓอᄎ൷ထৱຍࠄຑᥛሽᚘऱᣊֺॾᇆ࣋ՕΔྥ৵᠏ངګ։ᠦऱᑇڗ֏ᑇଖΖڇॾᇆ࣋Օࡉᑇڗ֏᠏ང৵Δൕ਩ۥาઽศ՗ऱ௽ۥᘛ٠ॾᇆ༉ᄎᆵࠩ௽ࡳऱຏሐΔڂۖւ๺։ᆢ։࣫Ζ
BD LSR IIሽ՗ߓอຝٙطሽᄭ൳ࠫΕຑ൷ᕴࡉࣨᑒխऱ๠෻ࣨΕࡉ෈ੌߓอ൳ࠫ໢ցΕگ࠷ਐقᗉ࿛ցٙࢬิګΖءີᆏႛ១࿍տฯڇInstrumentᏚᕴီ࿗Laser tab੒଄խΔڶ༓ଡᣂՊሼ୴Εױࠎࠌشृᓳᆏૹ૞೶ᑇΔڕՀቹDelayΕWindow ExtentionΕፖArea Scaling FactorsΙუᛵᇞڶᣂሽ՗ߓอऱݙᖞᇷಛΔױא೶ەπBD LSR II User’s GuideρխऱSignal detectionີᆏΖ
ຍ༓ଡᣂՊሼ୴ऱૹ૞೶ᑇᐙ᥼ᖞଡᏚᕴߓอሎ܂Ζԫ౳ऱس᠔ઔߒش֪լᏁ૞ޓޏLaser DelayፖWindow Extentionࠟႈቃ๻යٙΔᓮ֎ᖐ۞ޓޏΖ׽ڶ֟ᑇൣउՀױאڂᚨ௽௘าઽ؆ীΕ֡՚ΔࠐଥޏFSC Area ScalingΔ܀ᓮڇޢֲᣂᖲছΔᓮބڃDefault ଺ᐗ๻ࡳΔא܍ᐙ᥼הԳऱኔ᧭࿨࣠Ζ
ޢֲᣂᖲழΔຍࠄ೶ᑇᄎ۞೯ᚏژΔሶֲၲᖲழΔঞਢਤֲऱ່ึ๻ࡳΖאՀ១໢տฯᖙ܂଺ঞΔᓮጥ෻୉ီኔᎾᏁ૞۞۩๵ᒤΖ
1.4.1 Laser Delay ሼ୴࢏ᙈழ஁
੡Ա૾܅լٵሼ୴ၴขسᘛ٠ॾᇆհઌյեឫΔ༼೏ᛀྒྷᨋඕ৫ΔLSR IIආشم᧯़ၴᖿ࿇ߓอΖՈ༉ਢլᄎሼ୴ڇլٵழၴΔፖาઽศ՗ٌᄎΔࢬขسऱॾᇆ٦ნᖞܧ෼Ζڼԫ๻ૠऱᚌរޢԫ֭ሼ୴ࢬگႃᘛ٠ॾᇆΔאᗑمऱ٠᧜ᙁԵΔױ྇܅ઌյեឫΖԫ౳ऱ๻ૠਢא៴٠ᥲ୴੡t0Δࢬא׽ڶદۥΕ࿫ۥΕࡉ࿫؆ሼ୴ڶᏁ૞ᒔᎁሼ୴࢏ᙈழၴΖ
1.4.2 Window Extensionףᐈ࿗
Window gateψ࿗॰ωਢԫଡॾᇆ౧ᓢ೏࣍ࢳଖऱழၴ࿗ΙۖψWindow Extention (ףᐈ࿗)ωঞז।Ꮪᕴ๠෻౧ᓢॾᇆऱழၴਢܡ࢏९ΖലϙWindow Extentionϙᓳ۟Ϙ0ϙΔױڇ່ాऱழၴ࿗փ஢஦ࠩ౧ᓢ᧢֏Δ܀౒រਢຝٝ౧ᓢآ౏Եᗨ։ΖBD LSR II଺ᐗቃ๻ऱψףᐈ࿗ω੡7პઞΔܛ౧ᓢছ৵ٺ࢏९3.5პઞΔᔞش࣍0.2~12.0პۏՕ՛ऱาઽศ՗Ζԫ౳ऱ୅ࠂᣊ೯ढาઽઃᔞشΖ
ڕᏁྒྷၦՕ࣍20პۏऱาઽศ՗ᓮፖߓอጥ෻୉൷੓Δᔞ৫چ༼೏ψףᐈ࿗ωΖመ৫࣋Օψףᐈ࿗ωᄎࠌॾᇆᅸᐳΰelectronic abortsαऱᖲ෷ᏺףΔࠌᑌءੌຒ࠹ࠩওૻΖ
1.4.3 Area scaling factors ᓳᖞ૿ᗨངጩڂ՗
๺ڍڂైױ౨ᄎᐙ᥼ᘛ٠ॾᇆऱ૿ᗨAࡉ೏৫HऱઌኙֺଖΔڕาઽՕ՛Εᙫ෈ੌຒ࿛Ζ੡Աᒔঅޢԫଡ౧ᓢॾᇆऱAࡉHऱᑇଖઌᅝΔܛֺଖ࿛࣍1Δױאᓳᖞ૿ᗨངጩڂ՗ΖאFSCॾᇆऱ٠ሽԲᄕ᧯੡ࠏΔᓳᆏ”FSC Voltage”Δࠌᘛ٠პ෺ஆᆵ࣍FSC-Hऴֱቹࠥ৫પ100x1,000๠ΖᨠኘFSC-AፖFSC-Hऴֱቹհᘛ٠ൎ৫Δڕ࣠ઌٵΔঞլᏁᓳᖞ៴٠ሼ୴ऱ૿ᗨངጩڂ՗Ζڕ࣠FSC-AፖFSC-Hᘛ٠ൎ৫լઌٵΔ࣍”Instrument”ီ࿗խរᙇ”Laser”।௑ᓳᇢ”FSC Area Scaling”ΖڕFSC-A՛࣍FSC-Hհᘛ٠ൎ৫Δᓳ೏FSC Scaling FactorΙ֘հঞᓳ܅Ζ
ޢԫ֭ሼ୴ٺڶٺᗑمऱ૿ᗨངጩڂ՗ΔڕڶᏁ૞ױଡܑᓳᖞࢨᒔᎁΖڕՀΖ
1.5 BD LSR IIՠ܂ీ
ᇷறऱᛧ࠷ፖ։࣫Δא֗BD LSR IIᏚᕴऱ࿪Օຝ։פ౨Δ݁ਢLSR IIՠ܂ీՂΔطFACSDivaຌ᧯൳ࠫᖙ܂Ζ
ᇠՠ܂ీץਔאՀຝ։Κ
BD଺ᐗ๵௑಻ᆜऱPCሽᆰΰܶᄶቕΕ᝶ᒌα
Ёش࣍ᛧ࠷ᇷறऱՠ܂ీؘႊ಻ᆜڶԫଡPentium R IV ࢨਢޓ೏్ऱ๠෻ᕴΔڶԫଡ۟֟1GB ऱRAMΔԫଡ40GBऱױشऱ࿏጗़ၴΔא֗Windows
XP Professional (US-English)܂ᄐߓอΖ
17ٹᘛኟ
൑ۥٱ।ᖲ
BD FACSDiva ຌ᧯version 5.0 or above
Ёᇷறऱᛧ࠷ፖ։࣫
Ё෈ੌߓอऱ۞೯ඔ೯Δᣂຨࡉ৺ੑᑓڤ
BD FACSDiva Data Manager
Ёᇷறऱໂٝፖᚏژ
รԲີΕΕBD LSR IIൄ๵ᖙ܂
รԲີ
ᙫੌ෈ᙓ࿟
ᙓ࿟
2.1ךჄᙫੌ෈
৬ᤜޢֲၲ೯ᏚᕴছΔࠉՀ٨ޡᨏךჄᙫੌ෈ᙓ࿟Κ
ࣈՀףᚘᒵΰ෡ጸᒵαࡉᙫੌ෈ጥΔຏመڇᔞᅝऱ൷ᙰՂऱ८᥆ᚘථՂਊᚘΔࠀࢮದػۥ 1.
ຑ൷ᕴΖ
ലᙫ෈ᙓ࿟൅ֽࠩᑒΔࠀࢮದᚘԺຏଅ֞ࠐਜ਼௛
2
3.
ඝᠾື።Δ࠷Հື።ΔࠀףԵᔞၦऱFACSFlowᙫੌ෈Ζ
࣋ڃື።Δᒔঅࠡ႕ۥቾׂፖᙫ෈ᙓ࿟യٽΔྥ৵ඝጹ።՗ၞۯᆜΖ
4.
ૹᄅ൷ڃףᚘᒵࡉᙫੌ෈ጥΔط࣍൷ᙰլٵ๵௑Δ෻ᚨլᄎ൷ᙑΖ
5.
2.2ଙೈᐒ෈
୲ᗨપ16֒ΔႿᓄޗᔆΔאᖪۥጥᒵፖ׌ᖲઌຑΖ৬ᤜޢֲၲ೯ᖲᕴছΔࠉՀ٨ޡᨏଙೈᐒ෈Ζش֪ױאؾီ۷ጩᐒ෈࿟ऱ۾የၦΔࠐංጩᙫ෈ऱໍਲၦΖ
ࣈՀᐒ෈ጥᒵΰᖪۥጥᒵαΔຏመڇᔞᅝऱ൷ᙰՂऱ८᥆ᚘථՂਊᚘΔࠀࢮದػۥຑ൷ᕴΖ 1.
ඝᠾΔࠀࣈՀᐒ෈ᄨየႚტᤞ໴ᕴΖ
2.
ലᐒ෈࿟൅ֽࠩᑒΔඝၲ࿟።Δଙೈᐒ෈Ζ
3
ףપ500 mLዦػֽΖ
4.
5.
ඝڃື።Ζૹᄅ൷ڃᐒ෈ጥᒵࡉᐒ෈ᄨየႚტᤞ໴ᕴΖ
ՂᖲࠩԫתழΔΔךჄᙫੌ෈
2.3 Ղᖲࠩԫתழ
ڕ࣠׌ᖲբඔ೯Δ߷شؘ֪ᏁࠉՀ٨ޡᨏךჄᙫੌ෈Δאᝩ܍ጥሁխขس௛ऐΖ׌૞೴ܑڇ
Ꮑᒔᎁ׌ᖲڇStandbyणኪΔ׼؆լ૞ࣈՀᙫੌ෈ጥΔࠀڇچࣨՂ଺ۯᖙ܂Ζ
ࣈՀףᚘᒵΖ
1.
ࢮದᚘԺຏଅ֞ࠐਜ਼௛Ζ
2
ඝᠾື።Δ࠷Հື።ΔࠀףԵᔞၦऱFACSFlowᙫੌ෈Ζ
3.
࣋ڃື።Δᒔঅࠡ႕ۥቾׂፖᙫ෈ᙓ࿟യٽΔྥ৵ඝጹ።՗ၞۯᆜΖ
4.
ૹᄅ൷ڃףᚘᒵΖ
5.
2.4 ֊ངՂᑌᑓڤ(optional)
ൕ໢ጥՂᑌᑓڤ֊ང۟პ֞ᒌՂᑌᑓڤழΔᏁ૞ലHTSറشհၞᑌಾ؆ጥא֗ᛀ᧯ຑ൷ᕴᇘՂΔࠀല֊ང᝶֊ང۟პ֞ᒌᑓڤΖ
ฝၲHTSঅᥨ።Ζ
1.
ല֭ᐶਮؐฝΔ࠷Հᛀ᧯ጥΖ
2.
՛֨ඝՀၞᑌಾհ؆୚ጥ(DCM sleeve)Δࠀݔ࿳ᆜ࣋ᝩ܍ᙊ؈Ζ
3.
ലHTSറشհၞᑌಾঅᥨ୚(SIT protector)՛֨ٻՂ୚ԵΔං۟ືຝࠀ᠙ጹΖ
4.
൷ՂHTSᛀ᧯ຑ൷ᕴ(sample coupler):
5.
a. ലᛀ᧯ຑ൷ᕴ՛֨ٻՂංԵΔऴ۟ტࠩॴԺྤऄᥛං੡ַΖ
b. ᒔᎁᛀ᧯ຑ൷ᕴՀֱጥᒵਢܡڶށڴࢨᚘމΔૉڶΔᓮ՛֨ඈೈΖ
c. א໢֫ലᛀ᧯ຑ൷ᕴ֭ᐶ۰Δࠀش׼ԫೋ֫ലՂֱհඝၨඝጹΖ
ࣹრ: ᛀ᧯ຑ൷ᕴՂֱፖၞᑌಾঅᥨ୚ࢍጤհၴᚨڶԫ՛੄ၴሶΖڕآᨠኘࠩڼၴሶΔᓮല
ᛀ᧯ຑ൷ᕴ࠵ՀࠀૹᄅᇘݔΖ
ല֊ངᕴ֊ང۟პ֞ᒌՂᑌᑓڤΖ
6.
2.5 Ꮪᕴၲᖲ࿓ݧ
໢ጥՂᑌᑓڤ
2.5.1
1. ၲඔาઽᏚሽᄭΙၲඔሽᆰΖ
2. ᒔᎁᙫੌ෈࿟ڶԶ։የऱFACSFlowΔᒔኔ።ጹΔࠀᒔᎁጥሁຏዃΖ
3. ലᐒ෈ଙൾΔڇᐒ෈࿟խףԵપ500 c.c. ዦػֽΖ
4. ඈೈ෈ੌመៀᕴխऱ௛᧯Ζ
5. ڕ׽ࠌش໢ԫሼ୴ΔᏁᄊᖲԼն։ᤪΙڕࠌشᠨሼ୴ჸ಻દ٠ΔᏁᄊᖲ֥։ᤪΙڕࠌش
ᠨሼ୴ჸ಻UV٠ΔᏁᄊᖲքԼ։ᤪΖ
6. ച۩ PRIME פ౨ࠟڻΖངՂPBSጥΔHIGH RUN ࠟ։ᤪ৵ܛױၲࡨ։࣫ᑌ঴Ζ
Ղᑌᑓڤ
2.5.2პ֞ᒌ
პ֞ᒌՂᑌᑓڤ
ၲඔาઽᏚሽᄭΙၲඔHTSሽᄭΙၲඔሽᆰΖ
1.
ٵ໢ጥՂᑌᑓڤޡᨏ2-5Ζ
2.
ച۩ PRIME פ౨ࠟڻΖਊRUN bottonΔࠌߓอ๠࣍ՂᑌणኪΖ
3.
4. ၲඔ FACSDiva ຌ᧯Ζ
5. រᙇՠࠠ٨Ղհ HTS Prime
6. ৱ Prime ݙګ৵Δܛױၲࡨ։࣫ᑌ঴Ζ
2.6 ඈೈ෈ੌመៀᕴխ௛᧯
ԫ౳ۖߢΔᙫ෈መៀᕴᄎڶ௛᧯ڍਢطᙫ෈࿟ၒ೓ࢬᖄીΖຍଡംᠲᄎ֧ದऱฆൄ෼ွΔਢش֪ڇՂᖲழ޲઎ߠٚ۶ॾᇆڇᛧ࠷ᢄቹՂΔլᓵਢشչᏖگႃຒ৫ຟԫᑌΖش֪ؾီᛀ਷ױߠᙫੌ෈ᒵലਢ़ऱΖ٦גา઎࿇෼ᙫ෈መៀᕴᇙՈ޲ڶ෈᧯Ζ
ԫ؟࿇سՂ૪෼ွΔᓮܛࠥല࠷Հᛀ঴ΔངՂDIֽΔࠀല෈ੌߓอ࣋StandbyΖࠉᅃ2.3൸ࢬ૪ޡᨏૹᄅჄየᙫ෈දΖჄየ৵ΔᓮࠉՀ٨ਐقඈೈ௛᧯Ζ
ല෈ੌߓอ࣋RUNΖ
1.
᎘᎘ࣁؚ෈ੌመៀᕴΔല௛ऐ᎔۟መៀᕴືጤΖ
2.
3. ਊՀຑ൷࣍෈ੌመៀᕴՂհػۥਜ਼௛ጥືጤၲᣂΔࠌ௛ऐඈנΖലᄨੌऱᙫ෈ژ࣋ࠩᔞᅝ୲
ᕴΔ՛֨֎႖ᏴΖ
4. ᛀኘ෈ੌጥᒵΖڕڶ௛ऐΔലᏚᕴ׳ೡհ෈ੌጥሁᔚणၲᣂᓳᠾΔࠌ௛ऐፖᄨੌऱᙫ෈ژ࣋ࠩ
ᔞᅝ୲ᕴΔ՛֨֎႖ᏴΖΖ
5.ԫ؟ᙫ෈إൄੌ೯ழΔױ࠷Հᑌ঴ጥΔྥ৵ਊHigh ΕਊPrime Ζ
6.ૹ៿ޡᨏ5
7.אإൄֱڤPrime Ζ
8. HI RUN ն։ᤪΖ
2.7 ᏚᕴᏚᕴᣂᖲᣂᖲᣂᖲ࿓ݧ࿓ݧ
ᣂᖲছؘ૞೯܂Κ৺ੑၞᑌጥፖ؆୚ጥΔ߻ַၞᑌጥങႾΕࢨڶ਩றྲྀఎΖ
2.7.1໢ጥՂᑌᑓڤ
1. ࠷ 3 c.c. FACSClean Ղᑌ঴Ζ
2. ലᑌ঴֭਍ਮؐฝપ1։ᤪΔᨃᏚᕴऱట़ߓอࢼ࠷પ2 c.c. ऱ෈᧯Ζ
3. ലᑌ঴֭਍ਮڃإΔਊ HI RUN Δྥ৵ᨃ FACSClean ৺ੑጥሁ 5 ։ᤪΖ
4. ࠷ 2 c.c. FACSRinse Ղᑌ঴Δૹ៿Ղ૪ޡᨏ2-3Ζ
5. ࠷ 2 c.c. dH2O Ղᑌ঴Δૹ៿Ղ૪ޡᨏ2-3Ζ
6. ࣹრ່৵׽ఎપ1 c.c. dH2O ڇᇢጥխΖ
7. ֊ང۟ Standby אܐথሼ୴ΔStandby ն։ᤪ৵ᣂຨาઽᏚΖ
8. ᓮଙൾᐒ෈ΔࠀڃჄߩജၦऱዦػֽΖ
9. ലᙫੌ෈࿟ךჄ۟Զ։የΖ
10. ᣂຨ࿓ڤΙᣂຨሽᆰΖ
2.7.2პ֞ᒌპ֞ᒌՂᑌᑓڤՂᑌᑓڤ
৺ੑጥሁ:
1. Choose HTS> Clean
2. រᙇDaily Clean-96 well U-bottom template Δរᙇʳˢ˞Ζ
3. ຌ᧯נ෼ᒔᎁီ࿗ڕՀΚ
ᓮݙګՀ٨ᄷໂՠ܂Κ
3.1 ࣍ 96 well plate ՂΔᓮڇՀ٨ well խ෌ף FACSClean solution ֗ DI water Κ
3.2 ലHTS Ղ።ฝၲΔࠀലᄷໂړऱ96 well plate ᆜ࣍ plate holder ՂΖ
ࣹრ: Well A1 ᏁኙᏘ plate holder ׳ՂߡۯᆜΖ
3.3 ።ՂHTS Ղ።Δᒔᎁᖲᕴڇ “RUN” ऱᑓڤΖ
ˇˁʳរᙇ O ˞ΖᏚᕴݙګ৺ੑ࿓ݧ৵Δᄎנ෼Հ٨ီ࿗Ζ
6. រᙇ O ˞Δᇞೈ৺ੑ࿓ݧΖ
ࣹრ: ڕՂᑌᛀ᧯ܶڶacridine orange (AO) or propidium iodide (PI) ࿛⊺ᆈ৫ለ೏հ਩ᕪΔᓮࠉՀ।ࠉՀ।ᄷໂᄷໂᄷໂʳʳ˗ˡ˔ʳറش৺ੑᒌറش৺ੑᒌΔΔᙇᖗᙇᖗʳʳϘDaily Clean-96 well U-bottom DNA” template ࠉᅃՂ૪ޡᨏ৺ੑጥሁ૪ޡᨏ৺ੑጥሁˍˍ
Wells
Solution Volume (μl)A1-A4
BD FACSClean Solution 250 B1-B4
BD FACSRinse Solution 250 C1-C4DI Water 250
ᣂᖲ࿓ݧΚ
11. ਊStandby אܐথሼ୴ΔStandbyն։ᤪ৵ᣂຨาઽᏚΖ
12. ᓮଙൾᐒ෈ΔࠀڃჄߩജၦऱዦػֽΖ
13. ലᙫੌ෈࿟ךჄ۟Զ։የΖ
14. ᣂຨ࿓ڤΙᣂຨሽᆰΖ
รԿີΕΕᏚᕴ๻ᆜSurface Markers
รԿີ
ڇءີխΔ൞ലᖂ฾ڕ۶ၞ۩քۥ܍੽ᘛ٠։࣫հᏚᕴ๻ᆜΖڇ൞ಖᙕԫଡᑌ঴ऱᇷறհছΔؘႊ٣௅ᖕࢬࠌشऱᑌ঴ᣊীࡉᘛ٠਩றऱլٵΔኙ٠ሽ଍ᏺጥሽᚘଖΔᇖᚍଖࡉࢳଖ๻ᆜၞ۩່ᚌ֏๻ᆜΙᆖመ່ᚌ֏๻ᆜ৵Δլٵᘛ٠೶ᑇՀऱؾᑑาઽຟ౨։܉ڇቹݮࠥ৫ՂΖ
ඔ೯ߓอၞ۩঴ᔆ൳່ࠫᚌ֏๻ᆜಖᙕᇷற։࣫ᇷறᣂຨߓอ
ኙੌڤาઽᏚऱ່ᚌ֏๻ᆜץਔ༓ଡޡᨏΙ
1. ࿆ᙕၞԵBD FACSDivaຌ᧯
2. ᄅ৬ԫኔ᧭ Experiment
3. ᘛ٠೶ᑇᙇᖗ
4. ᎅࣔᛧ࠷פ౨᝶
5. ່ᚌ֏ੌڤาઽᏚऱ๻ᆜΰܶAutoCompα
6. ๻ૠGlobal Sheetᛧ࠷ठ૿
7. ಖᙕኔ᧭ᑇᖕ
8. ᙁנΕႪԵFCS֮ٙΕໂٝኔ᧭ᚾூ
ءᆏലᙞ૪ڕ۶ܓشϙInstrument Setting (Ꮪᕴ๻ᆜ)ϙפ౨ΔኙLSR IIᏚᕴၞ۩ 6 ۥ։࣫๻ᆜ່ᚌ֏Ζኙ൞ۖߢΔ൞ڇᖙ܂ຍࠄޡᨏழΔᓮጐױ౨ਊᅃႉݧၞ۩Ζ
3.1 ࿆ᙕၞԵBD FACSDiviaຌ᧯
1ၲඔᏚᕴ׌ሽᄭΖ׌ሽᄭጸۥ᝶ۯ࣍าઽᏚ׳ছֱΖ
2ၲඔሽᆰΖோ૿ုםᇆਢAdministratorΔയᒘ࿆ᙕ௃խᙁԵBDISΔਊENTERΖ
3ၲඔ BD FACSDiva ຌ᧯Ζڇຌ᧯࿆ᙕ௃խΔᙁԵࠌشृټጠ֗യᒘΖΰಖ൓٣ᇿጥ෻୉عᓮࠩ඄ᦞയᒘα
NOTICEΚ܃ऱࢬڶኔ᧭ᇷறຟല๯অఎڇ܃۞աऱ࿏጗़ၴᇙΔࠡהش֪լ౨ၞԵΖ
4ᒔࡳຌ᧯ፖาઽᏚݙګຑᒵΖݙګຑᒵ৵ΔڇInstrumentီ࿗Հֱᄎ᧩قInstrument ConnectedΖڕڶؘ૞ΔױរᚰInstrumentΰαီ࿗Δᨠኘຌ᧯ፖาઽᏚਢܡݙګຑᒵΖ
ՠ܂ီ࿗࿗ՠࠠ᥏
ՠ܂ီ
5ڕڶᏁ૞Δױ໢ᚰՠ܂ီ࿗ՠࠠ᥏Ղऱՠࠠቹقΰαࠐᛵᇞઌᚨऱ
໢ցิګΔڕψBrowser (ោᥦᕴ)ωΕψInstrument (Ꮪᕴ)ωΕψInspector (ᇷಛ਷઎)ωΕψWorkseet (ՠ܂ቹ)ωΕࡉψAcquisition DashboardωΖ
6ڇຌ᧯խՠ܂ழΔီ࿗ױ౨ᄎឆ៲ΖױࠩViewՀࢮפ౨᥏઎ਢܡࢬڶؘᏁՠࠠઃբၲඔΖڕսآߠ௽ࡳီ࿗ΔױشView>>Reset positionsڃ༚ຌ᧯ቃ๻ီ࿗ۯᆜΖࢨᠨᚰઌᚨီ࿗ᑑ፾Δലᇠီ࿗ฝࠩՂᐋΖ
3.2 ᄅ৬ԫኔ᧭Experiment
7տฯBrowser (ោᥦᕴ) ऱ໢ցิګΖ܃վ֚ऱኔ᧭ᄎڇោᥦᕴီ࿗شᐋ్ऱݮڤࠐิ៣ΔFolder()>>Experiment()>>Specimen()>>Tube()Ζ೶ەՀቹΖ
8૞ขسԫଡFolderΔ٣אᄶቕរᙇោᥦᕴီ࿗փറ᥆܃ऱ࿏጗Δྥ৵រᚰFolderᑑ፾Ζࢨਊᄶቕ׳᝶ᙇNew folderΖ
9૞ขسԫଡExperimentΔ٣אᄶቕរᙇ܃ᄅᏺऱFolderΔྥ৵រᚰExperimentᑑ፾ΖࢨൕᘛኟՂֱᙇᖗϘExperiment>>New Experimentϙࠐᄅᏺኔ᧭Ζࠀڇᙟ৵נ෼ऱψExperiment templatesωֱ௃խΔᙇشψBlank Experiment with Sample TubeωΖ
10” Blank Experiment with Sample Tube” ᚾ݈աܶڶԫSpecimen_001Δڕ࣠܃ऱኔ᧭ᚾ݈խ޲ڶSpecimenΔរᚰSpecimenᑑ፾ᄅᏺհΖ
11ޢଡSpecimenᚨ۟֟ڶԫᇢጥTube_001ΔױរᚰSpecimen_001ছψЀωᇆ୶ၲհΔࠌױߠΔڕՀቹΖޢଡ़ػTubeֱؐڶԫઌኙᚨψਐقᒢᙰωΰαΔ࿛ԫՀ։࣫ᑌ঴ழΔؘႊشᄶቕរᚰΔࠌࠡܧጸۥΰαΔթ౨ၞ۩Ꮪᕴ೶ᑇ๻ᆜΔܡঞ઎լࠩٚ۶Ꮪᕴ೶ᑇΖ
Ꮪᕴ೶ᑇ๻ᆜ
ᘛ٠೶ᑇᙇࡳΕΕܔ྇
3.3ᘛ٠೶ᑇᙇࡳ
12ڇInstrumentᏚᕴီ࿗ᄚΔ໢ᚰ”Parameters”଄૿Ζش֪ױڇParameters଄૿ՂΔܔೈٚ۶܃լࠌشऱ೶ᑇΖຍԫޡኙ૞ࠌش۞೯٠ᖂᇖᚍऱش֪ॺൄૹ૞Ζ
NOTiCEΚᓮᒔᎁشᄶቕរᚰTube_001ֱؐԫψਐقᒢᙰωΰαΔࠌࠡܧጸۥΰαΔթ౨ၞ۩Ꮪᕴ೶ᑇ๻ᆜΖ
13Parameters଄૿ڕՀΖ໢ᚰီ࿗ᄚऱϙAdd or Deleteϙ᝶ࠐᏺףࢨܔೈ೶ᑇΖڕᏁޓޏ೶ᑇΔױរᚰ೶ᑇֱ௑խ׳ೡᒢᙰΔڕFITCױޏګEGFPࢨAlexaʳ488ΖPerCP-Cy5.5ױޏګPerCPΖ
NOTICEΚݺଚ৬ᤜរᙇFSC-WፖSSC-WΔشࠐඈೈᠨᜤ᧯Ζ
14ݺଚڇڼᒭ฾૞شFITCΕPEΕPerCP-Cy5.5ΔPE-Cy7ΔAPCΔAPC-Cy7࿛քଡᘛ٠೶ᑇΖᏚᕴီ࿗ᄚ”Parameters”଄૿ᚨᇠڕՀቹΖ
3.4 ᛧ࠷൳ࠫ᝶
ᛧ࠷൳ࠫ᝶ΰΰAcquisition Dashboardα
15ᛧ࠷ᖙ᜕ิٙץܶᖙ᜕׌ᖲΕၞ۩ᛧ࠷ࡉಖᙕᑇᖕᇷறΖױਊՠ܂़ၴՂՠࠠၨխΰαΔࠐ᧩قᇠิٙΖ׽ڶᅝሽᆰՠ܂ీڶຑ൷ࠩาઽૠᑇᏚழΔᛧ࠷ᖙ᜕ิٙթ౨๯᧩قΖᛧ࠷ᖙ᜕ิٙפ౨ڕՀΚ
• Next TubeΚ։࣫Հԫጥᛀ঴Ζࢨᏺףԫጥᛀ঴Ζ
• Acquire DataΚᛧ࠷ΔၲࡨڇᘛኟՂ᧩قྒྷၦࠩऱาઽᇷறΙ܀ࠀլಖᙕΖ
• Record DataΚಖᙕΔၲࡨࠉਐחಖᙕᇷறګlist mode dataΖಖᙕݙګழΔᑌ঴ጥऱቹقᄎൕΰα᧢ګΰαΖ
• RestartΚڇAcquireࢨRecordᑓڤழΔ૞ޣխַૹྒྷΖ
• Storage G
ate Κᚏژ๻॰Δᄎᒔࡳୌࠄาઽᆢലץܶڇ܃ऱᑇᖕᚾூᇙΖຏൄ܃ᄎუ૞ఎՀ‘All
Events'ࢬڶࠃٙΙࠌشP1๻॰ঞ׽ᚏژP1าઽᆢΖ
• Stopping G
ate Κೖַ॰Δუᛧ࠷ԫ௽ࡳᑇၦऱาઽࠅᆢΔױࠌشP1ೖַ॰10Δ000ऱפ౨Δ
ലگႃ٤ຝࠃٙऱᑇᖕऴࠩP1 ሒࠩ10Δ000թೖַΖΖ
• Events to display Κຍפ౨ᒔࡳቹՂڇگ࠷ཚၴല਍ᥛ୶قڍ֟ଡࠃٙΖ
3.5 ່ᚌ֏Ꮪᕴ๻ᆜ່ᚌ֏Ꮪᕴ๻ᆜΰΰAuto-Compensation αNOTICE Κڕ࣠܃໛ᦟ֫೯ᇖᚍ܃ऱᑌ঴Δױאฃመຍଡ੄ᆵΖ
16ࠌش۞೯٠ᖂᇖᚍऱش֪Δ܃Ꮑ૞ᄷໂԫ֭޲਩ۥ़ػኙᅃΕࡉԫิൎ਩ۥऱ໢ۥኙᅃิΔא՛ቕැ֣෺੡ࠏΔܛCD4ΕB220ΕCD3ΕCD8Ε࿛࿛೏।ሒၦݼ଺ᑑಖΖ
17װᏚᕴᙇ໢ΔᙇᖗᏚᕴ๻ࡳΔࠀ׊ᙇᖗExperiment > Compensation Setup > Create Compensation Controls Ζ
18ڇWorksheet ီ࿗ᇙΔቃᆜ٠ᖂᇖᚍ૿ࣨലנ෼ԫ֭Unstained ़ػኙᅃΕࡉք֭܃ᙇᖗऱ໢ۥኙᅃิΖຍࠄWorksheet ।ࡉݺଚܛലࠌشࠐᛧ൓ᑌ঴ऱGlobal Sheet ९ቝڶࠄլٵΔۖ׊լױૹᒳ
Ζ
NOTICEΚڕ࣠܃޲ܔೈآࠌشऱ೶ᑇΔ܃ؘႊൕsectionte 3.3ૹᄅၲࡨΧ
19ଈ٣࣋Ղ़ػኙᅃิΔᓳᆏFSCࡉSSCሽᚘଖΔࠌ౨ജᔞᅝچ᧩قؾᑑาઽᆢΔ૞រڇࠌڶრᆠऱගᆢ౨ᗑمګᆢΔࠌ౨ڇ৵ᥛഎᙇழ൓ࠩۍ։ۍऱ໢ԫගᆢΰڕቹΔ٤ۨխԿଡػۨ෺าઽᆢαΖݙګ৵Δ٦ਊԫڻAcquire᝶ױխַᛧ࠷Ζ
20܃ലᏁ૞ڇཋ୴٠ቹᢜՂނP1 ॰ฝ೯ࠩ܃ऱؾᑑගᆢՂΖฝ೯P1 ॰৵ಖ൓ᄶቕ׳ᚰP1ࠀᙇᖗ‘Apply to all Compensation Tubes'Δױ᠙ࡳP1 ॰ऱᄅஆᑑΖ
21෼ڇױאᓳᖞ٠ሽ଍ᏺጥԱΖ໢ᚰ ”Instrumentϙီ࿗խऱϙParameterϙ଄૿Δᓳᖞٺଡ٠ሽ଍ᏺጥऱሽᚘଖΔࠌહནᘛ٠ଖᕣၦ၀२ؐՀߡΔࠀࠌาઽගᆢݙᖞܧ෼Ζݙګ৵Δ٦ڻ໢ᚰϘAcquisition Dashboardϙီ࿗ᇙऱϘAcquireϙ᝶ࠐխַᛧ࠷Ζ
22़ػኙᅃᑌءጥFITC-A vs PE-AቹݮΖ
ലອࢤኙᅃऱޢጟᘛ٠೶ᑇॾᇆ݁ᓳᖞڇรԫ్ኙᑇ೴഑փΰڕቹαΔڇڕLSR IIΕFACSCanto࿛ᖲীալٽழࡵΖ
23ڇլRecordऱൣݮՀΔດԫՂᑌք֭໢ۥኙᅃΖڕڶᔞᅝچዠࡳݼ᧯ᖺ৫Δ܃ᚨᇠ౨઎ߠԫଡၺࢤ୽Δ،ፖອࢤ୽۟֟૞։ၲԫଡኙᑇ్Ζ
24ݺଚ෼ڇաᆖױאᚏژࢬڶኙᅃᇢጥऱ଺ࡨᑇᖕԱΖᒔᎁោᥦᕴխΔጸۥऱᛧ࠷ਐقΰαۯ࣍ϘUnstained ControlϙΖ
25ಖᙕࠃٙऱቃ๻ᑇؾਢ5,000Δຍኙ࣍ૠጩ٠ᖂᇖᚍᚨբߩജΖ
26ଈ٣SIT࣋Ղ़ػኙᅃิΔ໢ᚰϙAcquisition Dashboardϙီ࿗ᄚऱϙRecordϙ᝶Ζݙګ৵Δຌ᧯ᄎ۞೯խַಖᙕΖ
27൷ထ໢ᚰϙAcquisition Dashboardϙီ࿗ᄚऱϙNext Tubeϙ᝶Ζ܃ᄎ઎ࠩԫଡFSC_vs_SSCऱቹΔ
ࡉԫଡڶP2ၴሶ॰ऱऴֱቹΔڕՀΖࠉݧՂᑌFITC໢ۥኙᅃิΔਊRecordΖ
28P2 ၴሶ॰ല۞೯װ༈ބ܃ऱၺࢤගᆢΖ
ڕ࣠ڶᏁ૞Δش֪ױאᓳᖞP2 ၴሶ॰ऱᒤ໮Ζ೶ەՀቹΖ
29ڇگႃݙԮ֭ኙᅃิᑇᖕΔࠀᒔࡳޢԫଡ໢ۥኙᅃิP2 ၴሶ॰ڶො።ၺࢤගᆢ৵ΔڃࠩExperiment > Compensation SetupᙇᖗCalculate CompensationΖ
30ຌ᧯ല૞ޣ܃ኙڼԫ٠ᖂᇖᚍఢೄࡎټΔ৬ᤜࠌشፖ܃ऱኔ᧭ઌٵऱټጠΖ܃ױאᙇᖗLink&Save ୚شڼ٠ᖂᇖᚍଖࠀᚏژאࠎֲ৵ࠌشΔࢨរᙇApply Only໢ొ୚ش࣍ڼڻኔ᧭ՂΖ
NOTICEΚݙګAutoCompensation٠ᖂᇖᚍ৵Δߓอലլւ๺܃ޏ᧢܃ऱᘛ٠ሽᚘΔլመΔ܃ױאޏ᧢FSC ࡉSSC ሽᚘΖ
ᓳᆏࢳଖ
30ԫ౳ش֪ৰࣹ֟რࢳଖऱ๻ᆜΖࢳଖऱ๻ᆜਢ੡ԱඈೈॺؾᑑॾᇆऱեឫΖڕڶᏁ૞ΔױڇՂᑌழΔ
໢ᚰ ”Instrument ϙီ࿗խऱϙThreshold ϙ଄૿Δࠀڇڼ๻ࡳFSC ࢳଖΖՀቹխױߠڇլٵࢳଖழΔڶயࠃٙऱֺ෷հլٵΖ
31װᏚᕴီ࿗ࠀរᚰ٠ᖂᇖᚍ੒଄Δؾছຍଡኔ᧭ऱ٠ᖂᇖᚍᑇଖലᄎ᧩قڇ߷ᇙΖ NOTICE ΚᓮᒔᎁڶشᄶቕរᚰTube_001ֱؐԫψਐقᒢᙰωΰ
αΔࠌࠡܧጸۥΰαΔթ౨઎ࠩᏚᕴ೶ᑇΖᒔᎁڶᘅ೯‘Enable Compensation' Ζ
32൞բݙګքۥ܍੽։࣫ऱᏚᕴ๻ᆜΖױ٨ٱϙExperiment ϙᚾ݈ՀऱᏚᕴ๻ᆜΖᄶቕ׳᝶໢ᚰኔ᧭
݈֮ٙխϙInstr Settings ϙቹقΔࠀڇInpector ီ࿗խΔᙇᖗϘPrint ϙ٨ٱ๻ᆜΔױ၀ࠩኔ᧭࿝ಖءխΖ3.6 ๻ૠGlobal Sheet ᛧ࠷ᒤء
33੡܃ܛലၞ۩ऱኔ᧭৬م܃ऱG lobal Sheet ठ૿Δଈ٣រᚰ Worksheet ૿ठؐՂߡհWorksheet
֊ང᝶֊ང۟Global Sheet 1Ζ
34ڇ़ػ”Global Sheet1”ՂΔ٣ᙇᖗՂֱϙDot Plot ϙՠࠠᙇႈΔࠀڇᛧ࠷ᒤءរᢄ፹ࠟଡཋរቹΖΰ1αFSC-A vs SSC-A ΰ2αFSC-A vs FSC-W Ζ
NOTICE Κუ૞ࡐࡳՕ՛Δ᎘រԫՀܛױΙٍױشᄶቕٚ۶ືរࠀ׊ࣆ࣋ࠐޏ᧢Օ՛Ζ35ױီኔ᧭Ꮑ૞ףᢄཋរቹΖ
36׳᝶໢ᚰቹݮ݄ᑑၗ೶ᑇՂΔױאൕؾᙕխᙇ࠷ࢨଥޏቹݮՂऱ݄ᑑ೶ᑇΖ
ᙇᖗᙇᖗᘛ٠೶ᑇࡉᘛ٠೶ᑇࡉᘛ٠೶ᑇࡉݼ᧯ݼ᧯ݼ᧯ᑑ᧘ᑑ᧘
37ࡎټᑌ঴ፖᇢጥټΖቃ๻ᙁנᚾټ੡Specimen_001_Tube_001Ζױࠉڼ଺ঞ੡൞ऱᑌ঴ߓอ֏ࡎټΖ
38אᄶቕរᙇSpecimen_001ΔࠀڇInspector ֱ
௃խᙁ
ԵᄅټΔڕSample_0831ΔਊEnter Ζ
39אᄶቕរᙇTube_001Δࠀᚨشᄶቕ׳᝶ޓټRenameפ౨Δྥ৵ᙁԵᄅټΔڕCD3_CD4_001ΔਊEnterΖࣹრΚDivaຌ᧯լױආش؁រᅝ೶ᑇټࢨᚾூټΖ
40ڇϙAcquisition Dashboardϙီ࿗ᄚਊϙNext Tubeϙ᝶Δࢬ൓ऱՀԫᇢጥല๯ࡎټ੡ᇢጥټ _ 002Δྥ৵ᇢጥ _ 003Δ࿛࿛Ζ
ᑑಖLabels
41אᄶቕរᙇTube_001ΔࠀInspectorڇϙLable (ᑑಖ)ϙ଄૿ՂΔ੡ٺଡᘛ٠೶ᑇᙁԵᑑಖڗցΖאᄶቕរᙇFITC೶ᑇ௑ΔڇLabel᥏ۯՂ᝶Եᑑق֮ڗΔڕCD3Ζࠉݧലࢬڶऱᘛ٠೶ᑇᑑقࣔػΖ
NOTICEΝຌ᧯ڇᑑ᧘ټᇙլւ๺ش՛ᑇរΖ
42ڇϙAcq. (ᛧ࠷)ϙ଄૿ՂΔױࠉኔ᧭Ꮑޣ۞۩๻ࡳψEvents to recordωΕψStopping gateωΕψStorage geteωΖ
܃ٍױڇࢬᏁtubesᄅᏺݙګ৵ΔរᙇExperiment > Experiment Layout ๻ࡳٺጥ೶ᑇټጠፖᛧ࠷යٙΖ
੡ؾᑑาઽᆢ੡ؾᑑาઽᆢഎᙇഎᙇഎᙇ๻॰๻॰
43ڇFACSDiva խ֧ၞၸᐋڤഎᙇΔቫᇢᨃഎᙇ॰ऱ৬مޓऴᤚΕ࣐ᚩΖլؘቝڇCellQuest ԫᑌ೚ؒ
ࣥڤሎጩऱ॰ΔቝਢR1*R2Ζ
ၸᐋڤഎᙇHierarchical G ating ऱഗء๻ૠΔਢA ஛॰խࢬขנऱ׼ԫ஛॰B ΔױאუګਢA ஛॰ऱԫଡऱ՛৘ΰ՗ႃαΖڇࠌشFACSDiva ຌ᧯ழΔᅝش֪ڇԫଡա๯എᙇࡳᆠመΔڕψ੒ऱาઽωऱቹᢜՂ٦ᒳᢄᄅऱ೴഑ࢨ॰Δຍᄅขנऱ॰۞೯ګ੡ψ੒ऱาઽω॰ऱ՛৘ΰ՗ႃαΖش֪ױאൕPopulation Hierarchial View פ౨խᨠኘࠩ܃ؾছࡳᆠנࠐࢬڶ॰ऱ୮ගᖫݮቹΖڕቹΖHierarchial View խ᧩قנψT cells ωΕψB cells ωΕፖψNK cells ωΔຟਢψlymohocytes ω॰ऱ՛৘ΖࢬאڇኔᎾՂ։ᙇᖙ܂ழΔش֪׽૞شψNK cells ω॰ΔהኔᎾՂਢࡳᆠڇψlymphocytes ٌႃNK cells ωΖ
44٣ᒔᎁោᥦᕴխΔጸۥऱᛧ࠷ਐقΰ
αۯ࣍ϘCD3_CD4_001ϙΙڇϙAcquisition Dashboard ϙီ
࿗խΔਊϘAcquire ϙΖ
45ڇGlobal Sheet1ՠ܂ቹՂΔᙇᖗڍߡݮഎᙇՠࠠΔࠀڇFSC-A vs SSC-A ቹՂᢄഎנψؾᑑาઽω
ۯᆜΔݮګψP1॰ωΖψP1॰ωᙇࡳ৵Δ॰փาઽᄎ۞೯ܧۥΖ൞ױאฝ೯،ΔࢨਢࣆڵϘ॰ϙՂऱٚ۶ԫ൳ࠫរΔࠐޏ᧢Ϙ॰ϙऱՕ՛ࡉݮणΖᒭ฾઎઎Ζ
46ՀԫଡቹᢜຏൄਢFSC-A_v_FSC-W Δຍଡቹᢜऱ܂شڇՊ೴ܑΕඈೈᠨᜤ᧯Ζشᄶቕ׳᝶໢ᚰᙇխFSC-A_v_FSC-W ቹᢜΔᙇᖗϙShow Population ϙࡎחΙ൷ထᙇᖗϘP1ϙΔڕቹΖຍᑌԫࠐΔࢬڶᙇխऱቹݮՂ༉׽᧩قP1ැ֣าઽᆢ᧯ΖΖ
47ՀԫޡਢڇFSC-A_v_FSC-Wቹᢜᢄഎנψ໢ԫาઽωۯᆜΔඈೈᠨᜤ᧯Ζڇ׌૞ऱาઽගᆢࡌ໮ࢮԫ஛॰ΔຍലਢP2ΖڕՀቹΖ
48൷ՀࠐΔݺଚუ૞شຍP2॰ࠐኙ٤ຝᘛ٠ቹᢜၞ۩ቹᙇΖਊ۰ϘShiftϙ᝶Δشᄶቕຑᥛរᙇࢬڶᘛ٠೶ᑇཋរቹΖشᄶቕ׳᝶໢ᚰᙇխࠡխԫଡቹݮΔᙇᖗϙShow Population ϙࡎחΙ൷ထᙇᖗϘP2ϙΔڕቹΖຍᑌԫࠐΔࢬڶᙇխऱቹݮՂ༉׽᧩قP2าઽᆢΖ
49ڕڶؘ૞ΔױڇڶᘋᔊऱቹݮՂף๻ගᆢ೴഑ΰpopulationsαΕອၺ੺ૻΰmarkerαΕࢨ؄ွૻΰquadrantsαΕ֗ઌᣂอૠ।ΔאঁᨠኘΖ
ڕ࣠უᨠ઎෼ڶऱගᆢၸᐋΔױאൕPopulation>>ᙇᖗShow Population HierarchyΖޢଡගᆢऱټጠױאشRenameפ౨ૹᄅᑑಖΔڕՀቹխCD4, CD8Ζ
50ڕ࣠უޏ᧢ٺଡගᆢऱቃ๻ᠱۥΔᠨᚰڇᇠගᆢছֱڶۥֱ௃ױᙇۥΔڕՂቹΖ
51ڇPopulation Hierarchyီ࿗ࢬ༼ࠎऱอૠڶΝٺଡගᆢऱᑇၦΕ،ଚ۾׀ئ॰ऱۍ։ֺΕࡉ،ଚ᜔ࠃٙऱۍ։ֺΖ
ڕ࣠უ૞ขנMeanΕCV࿛อૠΔאᄶቕរᚰٚრቹᢜΔࠀڇቹՂ़ػ๠Δ໢ᚰ׳᝶ᙇᖗψCreate statistics ViewωΖ
52ڕᏁᒳᙀอૠขנႈؾΔאᄶቕ׳ᚰอૠ।़ػ๠ΔᙇψEdit Statistics ViewωΖ
3.7 ಖᙕኔ᧭ᑇᖕ
53ലᇘڶ6ۥᘛ٠਩ۥऱSample_001ᑌ঴ጥᇘԵሉᑌകΖڇោᥦᕴխΔᒔᎁጸۥऱᛧ࠷ਐقΰαۯ࣍ϘCD3_CD4_001ϙΖױ٣໢ᚰϙAcquisition Dashboardϙီ࿗ՂऱϙAcquire ϙ᝶ΔৱาઽၲࡨڇቹݮՂנ෼ΔࠀᒔᎁP1/P2ۯᆜإᒔ৵Δ໢ᚰϙAcquisition Dashboardϙီ࿗ՂऱϙRecord ϙ᝶Ζ
54ಖᙕݙ10,000ଡาઽ৵Δലᑌ঴ጥൕሉᑌകՂ࠷ՀΖ
55ڇϙAcquisition Dashboardϙီ࿗Ղ໢ᚰϙNextϙΔᄅ৬รԲଡϙTubeϙΖࣹრΚڼழᄅ৬ऱϙTubeϙ۞೯๯ࡎټ੡ϘCD3_CD4_002ϙΖ
56ڇោᥦᕴխΔᒔᎁጸۥऱᛧ࠷ਐقΰαۯ࣍ϘCD3_CD4_002ϙΖലᇘڶรԲ֭ᑌ঴ጥᇘԵሉᑌകΖױ٣໢ᚰϙAcquisition Dashboardϙီ࿗ՂऱϙAcquire ϙ᝶ΔৱาઽၲࡨڇቹݮՂנ෼ΔࠀᒔᎁP1/P2ۯᆜإᒔ৵Δ٦໢ᚰϙRecord ϙ᝶ၲࡨಖᙕΖ
57ૹ៿Ղ૪։࣫ޡᨏΔ۟։࣫ݙࢬڶᛀ঴Ζ
֮ٙΕΕໂٝኔ᧭ᚾூ
3.8 ᙁנFCS֮ٙ
58݈֮ٙփऱᑇᖕᇷறຟ౨ജא“FCS֮ٙ”ऱݮڤᙁנΔຍጟ֮ٙݮڤ౨ജ๯BDຌ᧯ࡉࠡהᚨشຌ᧯ᦰ࠷Ζຍࠄ֮ٙױא“FCS 2.0”ࢨ“FCS 3.0”ठءݮڤᙁנΙՈ༉ਢᎅΔຍࠄᇷற౨ജא1,024ࢨ262,144ࠟጟᇞ࣫৫ᙁנΖڕ࣠൞ݦඨڇBD CellQuestΔBD CellQuest ProࢨModfit LTຌ᧯խૹᄅ։࣫ຍࠄᇷறΔ߷ᬕ൞Ꮑ૞ലᇷறא1,024ऱᇞ࣫৫ΰFCS 2.0αऱ௑ڤᙁנΖ
NOTICEΚڇBD CellQuest Proຌ᧯խ։࣫ᙁנऱᇷறհছΔଈ٣Ꮑ૞ࠌشBD FACSConvertຌ᧯Δല֮ٙ᠏ངګ౨๯Macintoshሽᆰᦰ࠷ऱ֮ٙ௑ڤΖ
NOTICEΚڕ࣠൞ᙁנ֮ٙਢ៭ԱڇFACSDivaࢨFlowJoຌ᧯խኙࠡૹᄅၞ۩։࣫Δ߷ᬕ൞ױאᙇᖗ“FCS 3.0”ठءΖࠌشຍጟᇞ࣫৫ࡉ֮ٙठءΔࢬڶऱ೶ᑇຟല๯অఎ଺ࡨᒵࢤ๻ᆜΖڇ
FACSDivaࢨFlowJoຌ᧯խΔױאലຍࠄॾᇆຏመኙᑇ᠏ངΔ٦אኙᑇݮڤ᧩قڇቹݮխΖ59
ڇោᥦᕴխΔᠨរᚰؚၲუᙁנऱኔ᧭݈֮ٙExperiment()Ζ
60ᙇᖗ“File>>Export>>Export FCS”ࡎחΖ
61ڇ”Export Parameter”࿗Ցխᙇᖗ “FCS 2.0”௑ڤΖࢨ“FCS 3.0”௑ڤΖ
62ڕڶؘ૞ΔױᙇNoneࠐᜍ՛ᇷறᚾΖ໢ᚰ“OK”Ζ
63ڕڶؘ૞Δױڇؾᙕሁஉ“࿏጗DΚ”ΔףՂᄅ৬ऱ݈֮ٙऱټڗΖ໢ᚰ”Save”ܛױΖ
64NOTICEΚ໢ᚰ”Details”Δ൞ױא਷ᔹڶᣂຍࠄᙁנ֮ٙऱᇡาᇷறΖ
65൞բᆖګפچലᇷறᙁנ۟“࿏጗DΚ”Ղऱ“BD Export”݈֮ٙխΔ܃ױאشᙟߪ጗ᓤ፹൅ߨΖࢨृ໢ᚰ”Start”Δᙇᖗ“Program >Hewlett-Packard>hp dvd writer>hp dvd writer”Ζᙇᖗ”HP Record Now”
ࡎחΔലᇷற᠏ᙕ۟CD٠጗ՂΖ
ร؄ີΕΕᏚᕴ๻ᆜDNA Analysis
ร؄ີ
ڇءີխΔ൞ലᖂ฾ڕ۶ၞ۩១࣐DNA։࣫հᏚᕴ๻ᆜ່ᚌ֏Ζڇ൞ಖᙕԫଡᑌ঴ऱᇷறհছΔؘႊ٣௅ᖕࢬࠌشऱᑌ঴ᣊীࡉᘛ٠਩றऱլٵΔኙ٠ሽ଍ᏺጥሽᚘଖΔᇖᚍଖࡉࢳଖ๻ᆜၞ۩່ᚌ֏๻ᆜΖᆖመ່ᚌ֏๻ᆜ৵Δլٵᘛ٠೶ᑇՀऱؾᑑาઽຟ౨։܉ڇቹݮࠥ৫ՂΖ
ඔ೯ߓอၞ۩঴ᔆ൳່ࠫᚌ֏๻ᆜಖᙕᇷற։࣫ᇷறᣂຨߓอ
ኙੌڤาઽᏚऱ່ᚌ֏๻ᆜץਔ༓ଡޡᨏΙ
࿆ᙕၞԵBD FACSDiviaຌ᧯
ᄅ৬ԫኔ᧭ DNA Analysis
ᘛ٠೶ᑇᙇᖗ
๻ૠGlobal Sheetᛧ࠷ᒤء
່ᚌ֏ੌڤาઽᏚऱ๻ᆜ
ಖᙕኔ᧭ᑇᖕ
ᙁנFCS֮ٙΔModFit LT։࣫
ءᆏലאൕࠃDNA໢ۥ։࣫੡ࠏΔᙞ૪ڕ۶ܓشϙInstrument Setting (Ꮪᕴ๻ᆜ)ϙפ౨ΔኙᇠᏚᕴၞ۩๻ᆜ່ᚌ֏Ζኙ൞ۖߢΔ൞ڇᖙ܂ຍࠄޡᨏழΔؘႊጐױ౨ਊᅃႉݧၞ۩Ζ
4.1 ࿆ᙕၞԵBD FACSDiviaຌ᧯
1 ၲඔ׌ሽᄭΖ׌ሽᄭጸۥ᝶ۯ࣍าઽᏚ׳ছֱΖ
2 ၲඔሽᆰΖோ૿ုםᇆਢAdministratorΔയᒘ࿆ᙕ௃խᙁԵBDISΔਊENTERΖ
3 ၲඔ BD FACSDiva ຌ᧯Ζڇຌ᧯࿆ᙕ௃խΔᙁԵࠌشृټጠ֗യᒘΖΰಖ൓٣ᇿጥ෻୉
عᓮࠩ඄ᦞയᒘΔࠏڕPIname_Yournameα
NOTICEΚ܃ऱࢬڶኔ᧭ᇷறຟല๯অఎڇ܃۞աऱ࿏጗़ၴᇙΔࠡהش֪լ౨ၞԵΖ
4 ᒔࡳຌ᧯ፖาઽᏚݙګຑᒵΖݙګຑᒵ৵ΔڇInstrumentီ࿗Հֱᄎ᧩قInstrument
ConnectedΖڕڶؘ૞ΔױរᚰInstrumentΰαီ࿗Δᨠኘຌ᧯ፖาઽᏚਢܡݙګຑᒵΖ
5 ᙇᖗϘInstrument>Instrument ConfigurationϙࡎחΔ਷઎෼ژᏚᕴ಻ᆜΖݺଚڇڼ૞ലDNA
։࣫ழᏁᏺף೶ᑇΔڕPIΕDAPIףࠩ෼ڶᏚᕴ಻ᆜխΖᓮ٣DuplicateԫᄅᏚᕴ಻ᆜΕאDNA_001ࢨࠡהזᇆࡎټհΔਊOK৵Ζ
6٦ڇParameterֱ௃խΔਊAddᏺף೶ᑇΔ৬ᤜPIشBlue (Laser)ΕC(Detector)ࠐೠྒྷΔڕՀቹΖݙګ৵ΔਊOKΖ
4.2 ᄅ৬ԫኔ᧭DNA Analysis
7૞ขسԫଡFolderΔ٣אᄶቕរᙇ܃ऱᇷறᑒΔྥ৵រᚰFolderᑑ፾Ζ
8૞ขسԫଡExperimentΔ٣אᄶቕរᙇ܃ᄅᏺऱFolderΔྥ৵រᚰExperimentᑑ፾ΖࢨൕᘛኟՂֱᙇᖗϘExperiment>>New Experimentϙࠐᄅᏺኔ᧭Ζࠀڇᙟ৵נ෼ऱψExperiment templatesωֱ௃խΔᙇشψBlank Experiment with Sample TubeωΖ
9ޓټפ౨ΖኙFACSDivaຌ᧯ቃ๻ऱᚾ݈ټጠΔઃױૹᄅࡎټΖڇោᥦᕴխᚨشᄶቕរᚰ”
Blank Experiment with…” ኔ᧭ᚾ݈Δྥ৵ڇInspectorֱ௃խᙁԵ”DNA_001”ڗցΔਊEnterࠐࡎټڼᄅ৬ऱኔ᧭ᚾ݈Ζ
10” DNA_001” ᚾ݈աܶڶԫSpecimen_001Δڕ࣠܃ऱኔ᧭ᚾ݈խ޲ڶSpecimenΔរᚰSpecimenᑑ፾ᄅᏺհΖ
ޢଡSpecimenᚨ۟֟ڶԫᇢጥTube_001ΔױរᚰSpecimen_001ছЀᇆ୶ၲΔࠌױߠΔڕՀቹΖޢଡ़ػTubeֱؐڶԫઌኙᚨψਐقᒢᙰωΔ࿛ԫՀ։࣫ᑌ঴ழΔؘႊشᄶቕរᚰΔࠌࠡܧጸۥΔթ౨ၞ۩Ꮪᕴ೶ᑇ๻ᆜΖ
4.3 ᘛ٠೶ᑇᙇᖗ
11ڇោᥦᕴխאᄶቕᙇᖗڼ”DNA_001”ᚾ݈Հऱ”Instr
Settings” (Ꮪᕴ๻ᆜ)Ζ໢ᚰ”Inspector”ီ࿗ᄚ
ऱ”Parameters”଄૿Ζش֪ױڇParameters଄૿
ՂΔװೈڍਲऱ೶ᑇΔݺଚڇڼᒭ฾װೈ
PerCP-Cy5-5ΔPE-Cy7ΔAPCΔAPC-Cy7࿛؄ଡᘛ
٠೶ᑇΔఎՀFITCࡉPEᘛ٠೶ᑇΖ
12 Parameters଄૿ڕ׳Ζ໢ᚰီ࿗ᄚऱϙAdd or Deleteϙ᝶ࠐᏺףࢨܔೈ೶ᑇΖڕᏁޓޏ
೶ᑇΔױរᚰ೶ᑇֱ௑խ׳ೡᒢᙰΔڕPerCP-Cy5-5ױޏګPerCPࢨPIΖ
13ڇڼ๻ࡳPI੡LINܧ෼ֱڤΔࠀᏺףPI-W೶ᑇΔڕՀቹΖ
4.4 ๻ૠGlobal Sheetᛧ࠷ᒤء
14ڇ़ػ”G lobal Sheet1”ՂΔ٣ᙇᖗϙDot PlotϙՠࠠᙇႈΔࠀڇᛧ࠷ᒤءរᢄ፹ࠟଡཋរቹΖΰ1αFSC-A vs SSC-Aΰ2αPI-W vs PI-AΔᙇᖗϙHistogramϙՠࠠΔࠀڇᒤءរᢄ፹ԫଡཋរቹΰ3αPI-A histogramΖ׳᝶໢ᚰቹݮ݄ᑑၗΔױאଥޏቹݮՂऱ݄ᑑ೶ᑇΖ
4.5 ່ᚌ֏Ꮪᕴ๻ᆜ
່ᚌ֏Ꮪᕴ๻ᆜΰΰOptimize Settingsα
ءᆏխΔലຏመאՀնଡޡᨏΔᖂ฾Ꮪᕴऱ່ᚌ֏๻ᆜΖ
ᓳᆏࢳଖΔ๻ᆜPI=5,000
੡௽ࡳऱDNAᘛ٠೶ᑇᓳᆏ٠ሽ଍ᏺጥሽᚘଖ
੡໢าઽᆢ᧯๻॰
ᓳᆏFSCࡉSSC๻ᆜ
ࠡהᘛ٠਩ற೶ᑇᓳᆏ٠ሽ଍ᏺጥሽᚘଖΰoptionalᠨۥα
אൕࠃDNAᠨۥ։࣫੡ࠏΔലࠌشאՀᘛ٠਩றኙᅃᑌ঴ጥΚΰ1αUnstimulatedኙᅃΔPI ໢਩Δΰ2αStimulated PositiveኙᅃΔPI໢਩Δΰ3αStimulated PositiveኙᅃΔPI and Anti-H3 FITC ᠨ਩Ζ
ެࡳ๻ࢳ೶ᑇPI
15٣ᒔᎁោᥦᕴխΔጸۥऱᛧ࠷ਐقΰαۯ࣍ϘTube_001ϙΙ໢ᚰ ”Instrumentϙီ࿗խऱϙThresholdϙ଄૿Ζڇڼ๻ࡳࢳଖ೶ᑇPIΔࠀᓳᆏPIࢳଖ5,000Ζࢳଖऱ๻ᆜਢ੡ԱඈೈॺؾᑑॾᇆऱიឫΖ
16DNA։࣫ආLowੌຒΖ
17ലອࢤኙᅃጥᇘԵሉᑌകΖ໢ᚰϙAcquisition Dashboardϙီ࿗ᄚऱϙAcquireϙ᝶Ζڼਐחᄎࠌຌ᧯ܛழܧ෼Ꮪᕴࢬྒྷ൓ऱาઽᇷறΔ܀լ܂ಖᙕΰRecordαΖᓳᆏPIሽᚘଖVoltageΔࠌG0าઽऱPI-Aଖપڇ50ΰX1000αۯᆜΖ
੡໢าઽᆢ᧯๻॰
18ڇGlobal Sheet1ՠ܂ቹՂΔᙇᖗڍߡݮഎᙇՠࠠΔࠀڇPI-W vs PI-AቹՂᢄഎנψSingle CellsωۯᆜΔݮګψP1॰ωΖψP1॰ωᙇࡳ৵Δ॰փาઽᄎ۞೯ܧۥΖ൞ױאฝ೯،ΔࢨਢࣆڵϘ॰ϙՂऱٚ۶ԫ൳ࠫរΔࠐޏ᧢Ϙ॰ϙऱՕ՛ࡉݮणΖᒭ฾઎઎Ζ
ᓳᖞFSCፖSSCሽᚘ
19໢ᚰ ”Instrumentϙီ࿗խऱϙParametersϙ଄૿ΖᓳᆏFSCࡉSSCሽᚘଖΔຏൄ٣ᓳᆏFSCΔ٦ᓳᆏSSCΖᓳᆏFSCࡉSSCሽᚘଖVoltageΔࠌJurkat໢าઽගᆢΰܛદۥาઽᆢα౨ᗑمګᆢΔڕՀቹΖݙګ৵Δ٦ਊԫڻAcquire᝶ױխַᛧ࠷Ζ
20شᄶቕរᙇPI-AऴֱቹΖਊᄶቕ׳᝶ᙇᖗϙShow Population ϙࡎחΙ൷ထᙇᖗϘP1ϙΔڕቹΖຍᑌԫࠐΔPI-AऴֱቹݮՂ༉׽᧩قP1 Jurkat໢าઽගᆢΖ
21პᓳᆏࢳଖΖ໢ᚰϘAcquisition DashboardϘီ࿗ᇙऱAcquire᝶ΖᏁ૞ழΔ໢ᚰ ”Instrumentϙီ࿗խऱϙThresholdϙ଄૿Ζڇڼᔞᅝچ༼೏PIࢳଖΔאඈೈาઽᅷׂऱեឫΖ
ᓳᆏFITCᘛ٠٠ሽ଍ᏺጥ(optional for FITC/PI)
22൞ױאڇሎ۩ψ़ػኙᅃጥωழΔᓳᖞڇϙInstumentϙီ࿗ऱ٠ሽ଍ᏺጥሽᚘଖVoltageΔאᛀ਷FITC۞࿇ᘛ٠Ζല़ػኙᅃጥᇘԵሉᑌകΖ٣ᒔᎁោᥦᕴխΔጸۥऱᛧ࠷ਐقΰαۯ࣍ϘTube_001ϙ໢ᚰϙAcquisition Dashboardϙီ࿗ᄚऱϙAcquireϙ᝶Ζ
23Ꮑ૞ழΔ໢ᚰ ”Instrumentϙီ࿗խऱϙParameterϙ଄૿ΖᓳᖞFITC٠ሽ଍ᏺጥऱሽᚘଖΔࠌ൓ອࢤาઽऱFITCᘛ٠೶ᑇॾᇆ᧩قڇรԫ్ኙᑇ೴഑փΰڕቹαΖݙګ৵Δ٦ڻ໢ᚰϘAcquisition Dashboardϙီ࿗ᇙऱϘAcquireϙ᝶ࠐխַᛧ࠷Ζ
24൞բݙګ១࣐DNAᠨۥ։࣫ऱᏚᕴ๻ᆜΖױ٨ٱϙExperiment ϙᚾ݈ՀऱᏚᕴ๻ᆜΖ׳᝶໢ᚰኔ᧭݈֮ٙխϙInstr SettingsϙቹقΔࠀڇInpectorီ࿗խΔᙇᖗϘPrintϙ٨ٱ๻ᆜΔױ၀ࠩኔ᧭࿝ಖءխΖ
4.6 ಖᙕኔ᧭ᑇᖕ
ᄷໂՠ܂
25ࡎټᑌ঴ፖᇢጥټΖቃ๻ᙁנᚾټ੡Specimen_001_Tube_001Ζױࠉڼ଺ঞ੡൞ऱᑌ঴ߓอ֏ࡎټΖ
אᄶቕរᙇSpecimen_001ΔࠀڇInspectorֱ௃խᙁԵᄅټΔڕSample_0831ΔਊEnterΖ
26ٵ෻ΔאᄶቕរᙇTube_001ΔࠀڇInspectorֱ௃խᙁԵᄅNameټΔڕAntiH3_PI_001ΔਊEnterΖࣹრΚDivaຌ᧯լױආش՛ᑇរᅝᚾټΖ
27 ᑑಖLabelsΖᙇᖗϘExperiment>Experiment LayoutϙࡎחΖڇϙExperiment Layoutϙ
ီ࿗ՂΔ൞ױא๻ᆜ೶ᑇټጠΔڕՀቹΖຍࠄټጠലנ෼ڇቹݮऱ݄ᑑၗΔא֗ࢬڶอૠ࿨࣠խΖڇϙLable (ᑑಖ)ϙ଄૿ՂΔ੡ٺଡᘛ٠೶ᑇᙁԵᑑಖڗցΖאᄶቕរᙇFITC೶ᑇ௑ΔڇLabel᥏ۯՂ᝶Եᑑق֮ڗΔڕAnti_Histone H3Ζࠉݧലࢬڶऱᘛ٠೶ᑇᑑقࣔػΖ
28 ڇϙExperiment Layoutϙီ࿗ᇙϙAcquisition (ᛧ࠷)ϙ଄૿ՂΔ੡ϘCD3_CD4_001ϙආ
ႃጥΔެࡳಖᙕาઽ᜔ᑇΖڇϙEvents to RecordϙऱՀࢮפ౨।խᙇᖗϘ20,000ϙΔࢨ֫ՠ᝶Ե௽ࡳᑇڗ৵Δ໢ᚰϘOKϙΖ
29 ڇAcquisitionDashboardီ࿗ΔᒔᎁආشLowੌຒΖࠀࠉኔ᧭Ꮑޣ۞۩ᙇࡳψStopping
gateω֗ψStorage geteωΖ
ၲࡨಖᙕᇷற
30ᇷறᛧ࠷መ࿓խΔᓮᨠኘGlobal sheet1խPI-W/PI-AቹݮP1॰എᙇᔞᅝፖܡΖڕڶؘ૞Δױฝ೯P1॰ۯᆜΖ
31 ലᇘڶAnti-H3 FITC/PIᘛ٠਩ۥऱSample_001ᑌ঴ጥᇘԵሉᑌകΖڇោᥦᕴխΔᒔᎁ
ጸۥऱᛧ࠷ਐقΰαۯ࣍ϘAntiH3_PI_001ϙΖױ٣໢ᚰϙAcquisition Dashboardϙီ࿗ՂऱϙAcquire ϙ᝶ΔৱาઽၲࡨڇቹݮՂנ෼ΔࠀᒔᎁP1ۯᆜإᒔ৵Δ໢ᚰϙAcquisition Dashboardϙီ࿗ՂऱϙRecord ϙ᝶Ζ
ಖᙕݙ20,000ଡาઽ৵Δലᑌ঴ጥൕሉᑌകՂ࠷ՀΖ
32 ۞೯֒ᕨᇢጥټΖڇϙAcquisition Dashboardϙီ࿗Ղ໢ᚰϙNextϙΔᄅ৬รԲଡϙ
TubeϙΖࣹრΚڼழᄅ৬ऱϙTubeϙ۞೯๯ࡎټ੡ϘAntiH3_PI_002ϙΖ
ڇោᥦᕴխΔᒔᎁጸۥऱᛧ࠷ਐقΰαۯ࣍ϘAntiH3_PI_002ϙΖലรԲଡᑌ঴ጥᇘԵሉᑌകΖױ٣໢ᚰϙAcquisition Dashboardϙီ࿗ՂऱϙAcquire ϙ᝶ΔৱาઽၲࡨڇቹݮՂנ෼ΔࠀᒔᎁP1ۯᆜإᒔ৵Δ٦໢ᚰϙRecord ϙ᝶ၲࡨಖᙕΖ
33 ૹ៿Ղ૪։࣫ޡᨏΔ۟։࣫ݙࢬڶᛀ঴Ζ。