BD FACSCanto II
Transcription
BD FACSCanto II
BD FACSCanto II http://www.bd.com/tw/ Tel : 02-27225660 BD FACSCanto II BD FACSCanto …………… 03 BD FACSCanto Surface Markers……………….. 22 DNA Analysis..…………………. 34 FACSDiva ………………….………… 58 ! " # ………… 13 …………………… 47 2 BD FACSCanto II BD FACSCanto II • 488nm 20mw • 633nm 17mw + , - : ;< 6 9 DE 6~8 ! 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E 1.6 BD FACSCanto II ¦ § B ¿ FACSCanto II Z â Z ± X t L < PC h É ¦ § ¨ B Z 1 ² ³ L ¯ ® ° º « ÷ ª D U ¥ « V Y Z % C# PentiumR IV 0« É 1GB RAM# 40GB µ Windows XP Professional (US-English)Z · ` 17 ¸ © ¾ C#Ö a ¶ * S & ´ s J r w ¹ .û » BD FACSCanto II Clinical Software version 2.1 or above Instrument QC, Lymphocyte subset TBNK, HLA-B27, Stem Cell ± µ« BD ; BD FACSCanto II > w ² BD FACSCanto II Clinical or FACSDiva P r ¬ r Enumeration BD FACSDiva P ± ¦ ± 9: version 5.0 or above D § ¿ ` M B a N v N ú~ O î ¼ 5 BD FACSDiva Data Manager ± ¦ B : ¿½ Ô 14 BD FACSCanto II 2.1 5 F G H 1. F I J K¥ } ¾ ¿ À Á 2. F I 5 6 K h8_Ü X 3. BDIS (` ® > p Z a ª )Enter à 4. F I BD FACSDiva L K¥À Ï Á Ä 5. M N L O P Q A R K C² ¿ > 6. T r Y « Z r FACSFlowÑ g 9 µn Ê K ¥ Instrument É \ Ç S ü Å w Æ ¾ Instrument È ¿ %É Ë Ê P Ì D ¥InstrumentÉ ~ [ T ® Ê µn Î É lInstrument ConnectedSS ©Í Ê µn Í ©Í l ö Î Ï 9R & ) É T U | lInstrument DisconnectedÒ V W B Ð Î X y Ó \ v Ñ N FACSDivaP D 7. : Cytometer > Fluidics Startup 9: v N 5 ©¢ OK > ®Ô c Shutdown solution É Prime -- Bubble filter purge –Degas flow cell%Ô B BD FACSCanto II Ê Sheath buffer System 5~10Õ 15 N D E Ç 5 Ç Ö Instrument > Automatic Cleaning Þ Ö ~ \ Ó FACSDivaP D N v > FACSDivav N { N u©M Automatic Clean ] F I T 9: N ~ v N M 9: v 5 Automatic Clean ^ _ 8. ` I ' H P Q a bc d OKK D 9. S : For Diva 6.0 r E T + , Y Z e G P warmup time¾7:27Ú × ^ Q K X û T ~ Ú û É Z ð Î µn ! I¥ Instrument É Ê µn ©ÛÜ 10. f g h i j F k / l m n o pq r 5 s KÝ ^ µn Ê ©Í ` a Ù | v N l – Remaining lThe system is ready Í 9: Ø Þ ß à á 16 2.2 5 T t u v w x y z { | (Controls in Instrument Frame) Fluid Level Indicators . 5 BD FACSDiva P D <=9D 20%ä ©<=g lÌå æ & 4 • é R ç5 • D 5 I ¹ Ñ í © } Im X û + } ~ B > l É ª Ê FACSFlow ð µ& 5 ë × õ « 9Ù 5 l 9í î © ¥9R D Câ ã 4 X µº çè 0 ì o l l X ( 9í Ô S )0S <17%% 0ü 4 J ( U V ò í )è í ó ô Ñ >83%% X ¾ ' lÑ 9D ê p ñ 9D 95 95 0Ñ g #> 9ö b ` a FACSFlow í ¯ ©M N ù Iú~ ¡ú [ T ¢ S 0%%0ü B m 5 lÑ õ 0Ñ g ¯ b ü Laser Tab ý ; Iâ Y IÌ FACSFlow b ° þ I Window Extension ¿ 1 FSC Area Scaling ø 0@ ã l FACSFlow f >99%% ø : ÷ 9D #í lï ] 9n , 5 e¥q 5 l FACSFlow • ¥û ò o l17%34%51%68%… % æ shutdown cleaning b 5 5 j k z J ¤ BD FACSCanto II É $ ½u 17 Status Tab } ~ CB Ë Ì ! ©C¿ ý þ ÷ ! & ' ¦ B 2.3 x y Acquisition Dashboard § Y % Þ l§ Y Í ¼½ lâ 1 Y ¼½ ¼½÷ û _ CÌh @ § Z Cr I J ¦ B " > Z I§ J S Y _® ¼½ä ³ Í µ¾ • Next Tube¾ µ • Acquire Data¾§ • Record Data¾ • Restart¾¥ Acquire 0 Record ¼ • SIT Flush¾ N " Á s Á s SIT O ¥ î m 5 r Í ¹ Á ¦ B I 5 4 2 l ~ ¦ B Pã Á list mode data ® ] J À Ó A 18 Flow Rate Y § ¼½®Ö à : Low¾ 1 v ] U v : ] V ] : v ¾ ¾60 µl /min v : U ¯ ¾10 µl /min : Medium¾] High: V ¾120 µl /min v 2.4 G S H 1. « Ù 2. 9: d Fluidics Startup @ ; ` a Ò " « 55~65 Psi @ 6 3. ) Ö 1X10 cells/ml 4. « Ù ¢ o 5. « Ù d - · ß 6. \ 9R 5 l« 7. « Ù ¢ à #9D 8. eõ S À FALCON 2052 Î Ö g CY ® Ù L 2 ¬ ®Ò 0.5 ml B ®Î « C 9 « S ¢ Ù 9Ñ #N Z ©¢ ® ®$ . 2.5 5 ^ G H S T > ~ e @ Fluidics Shutdown BD FACS Shutdown solution0 % ¯ 5 r ! P" # % 9: É Ê 1. 3 ml FACSClean10%Bleach%] 2. ¥ Basic controls Acquire Data 3. & > Q ¥ High Flow Rate ¼ 5 µAcquire 5 Õ5 Õ~ ÛÜ Acquire r ® 4. µ FACSClean Ê BD FACSCanto II 3 ml dH2O ] 19 5. ¥ Acquisition controls Acquire& > ¥ High Flow Rate ¼ µAcquire 5 5 Õ 6. 5 Õ~ ÛÜ D E Acquire r bh ® µ] Q Fluidics Shutdown bc K 7. ^ _ ' Ç È ' Cytometer > Fluidics Shutdown' ' X Ø É ' T ~ M Ç D E Ù T N ®Ç Ê ' OK' Ö ' OK' r Ö s ú~ c M m D E ^ G a M D E fÙ N k ) * + b w à ) / , 0 9. ú~ > ú~ h # 7 10. O g | ( ) Õ FACSDivaP D ú~ J > u© Fluidics Shutdown P Q a b ^ _ q J K E r ú~ 5 D 8. 9: Automatic Cleaning ÷ v 9: 1 ml z ¡ h ' dH2O' . ] q C² ( ] + ) " # e ] $ P. SITK % 1/10 D ç5 ' M 1 6 ç¥] ÛÑ 2 9" 4 ' # 3 $ ò K Condensation Trap J Q ª 20 2.6 FACSCanto II ¢ £ Clean Flow Cell.. ¤ ¥ ' ( Ì > ( ) µ ] 3 mL FACSClean O I FACSClean O c 9m 9 95 c c ¥: N Instrument/Clean Modes/Clean Flow Cell..÷ P @ 8 O î ¿] 9? @ ª Õ : N ¡ ¿] @ ¥q 5 T ~ fluidics startup 0 fluidics shutdown ÷ BD FACSCanto II 21 De-Gas or Bubble Filter Purge ¤ ¦ § ¨ © 5 ª § ¨ Ò L d - X ¬ ¬ $ CV < } Ü 9ô 9 : ÒD å ë ¿ ù & = ; : Ò I i L , N J × I6 t > q ? B ÷ à g ¥O Ò L g A C: ¦ ± © 2 Ò ã ÒD û I # @ Instrument > Cleaning Modes > De-gas Flow Cell..÷ @ Instrument > Cleaning Modes > Bubble Filter Purge..÷ Priming « ¥ Í S : q ÷ ®þ CÒ @ ÂÖ C + ̽ ¬ 9: Y F PÑ G ; Z A 9ô ½ ~ 0 Ú t ²² ³ ù IB Ê 9 ½ I T ¯ { î 9 O 9 } Ï Ä (Long Clean) 9ô Ó OK 9ô IE S ý @ Long Clean ÷ O | \ Instrument > Cleaning Modes > Prime After Tank Refill..÷ P¥ä ~ S ¥Ê H ~ 0 9: CY E > c ; @ 5 ª « ¬ : D E r FACS shutdown b { 2 9: ; 9ø FACSClean~ # FACSClean>275 mL% ¿ 5 Ô B 75 T Û >1,100 mL% Instrument > Cleaning Modes > Long Clean..÷ © Ô 70 Õ è 22 2.7 FACSCanto II ® ¯ ¤ ° V 5 ± 9 ² ³ ´ 2 BD FACS 7-color setup I / @ Cat. No. 335775 I / @ Cat. No. 556288 Nile Red fluorescent particle I / @ Cat. No. 347240 beads Rainbow fluorescent particles µ ® ¶ · BD FACSFlow sheath fluid I / @ Cat. No. 342003 BD FACS cleaning solution I / @ Cat. No. 340345 BD FACS shutdown solution* I / @ Cat. No. 334224 House hold bleach for waste ¸ W 5mL polystyrene test tubes (BD Falcon™) uncapped, 125 per bag eJ BD Cat. No. 352052 capped, 125 per bag eJ BD Cat. No. 352054 capped, 25 per bag eJ BD Cat. No. 352058 with cell-strainer cap eJ BD Cat. No. 352235 *BD FACS shutdown solution BD FACSCanto II r " # ®K % ¿1 ç 23 ! 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Specimen_001 C ¡ | Ð ÇÈ Specimen_001 y a5 " l É z b G K µ S s ] I² 3.3 : ¾ d ¿ 9 ¥n o ®r È”Inspector”É ! "~ ¥q ® ! ! " PerCP-Cy5-5 3.4 "n ÇÈ! ¥ Parameters þRß # g ) p É } i ! " " Parameters þRÎ pÈÉ µ ”Instr Settings” ( > PerCP-Cy5-5PE-Cy7APCAPC-Cy7 G g Lß µ FITC PE c t ”Parameters”þRÍ | Ê ”2-color exp” s q ÖC < ®Î \Add or Delete\¯Þ | Ê Z z K FITC Q 0 g ! "B * EGFP 0 Alexa 488 PerCP 0 PI Global Sheet À ¡ BD FACSCanto II 27 10 ”Global Sheet1”E ¥S 6 # $ º Öö P 1%FSC-A vs SSC-A2%FITC-A vs PE-AΠǺ \Dot Plot\ n ÖC 6 ! ¥§ ¯pȺ ^Ç X 6 = , r " 3.5 Á  5 r Optimize Settings rì ^G ®¢Ø G G R < É S 6 % Ä 11 # ( FSC SSC { N _ @ D y A 0 ) ò L> NO É É ] ! B " G h P h Q < z %CD3 FITC p ¢Srµ ì 3%CD4 PE p Ï Ï A B Ï ] ¾ %S 6 FSC @ SSC r E 12 rµ Surface Markers M Y Ï Ã Ù r¥? ¢S © Ø 6 SP n @ o Ï D vS ®æ ¤ u Ï 6 I{ § } \I > ] A 5 l ¥\Instument\É ø %Ü ¦ B # þRk ú É vTube_001\ X pÈ\Basic Controls\É T Ê ã Z Á Ê | \Acquire\¯q 5 Record% 28 FSC SSC h G Ç¥SC R Ä Å 13 7 § 14 · Í [ l ®à <Ø E @ FSCÛ G G # º ®\Parameters\þ Ê SSCT ~ÛÜ Æ pÈvBasic ControlsvÉ 6 S ³ Ì % pÈ ”Instrument\É + Threshold\þR¥q 8 FSC SSC h G Acquire ¯ ® à < VoltageS Ç È Ã N × 0« { R "P <! IpÈ ”Instrument\É FSC R G <R É < « Ê ®\ g õ É ¥ Global Sheet1 a Acquire ¯B ¦ Ê É bÜ Z º ÖC P Ö aP1 ]b aP1 ]bÖ{ v]\@ BD FACSCanto II i AU V ÇÞ ¥ FSC-A vs SSC-A º ~]ª v]\© d © M N { ! L\ \ r 29 15 vShift\¯ # º 6 Ê Ë Ö®º Ã Ä : r¥? @ Í ¢S Ï 6 ü C ÇÖA P1 l D } > aS Ï 6 ¤ bI <ST T # ] E Ï 6 S ¦ B ø a . ¥º rè ÖC ® Î ® ¯pÈÖ®U vP1\º ¥\ Instument \É ¯ ø IË Ê n Ø Z Ì ®æ § ! 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" 19 j Ø Ù Ú Û Ü Ý Þ " #Ic ¨ j 5 Iv ] â I 20 dD Ì R ° ã ä 2 á » B à © IGN B C * ä ß ^ à ! ± á B C GP1 >H á P 6 Color LnLIØ Æ G Inspector z hIdhAnalysisi õ 21 Z | } ~ 6 Color LnLIØ Æ dhBatch Analysisi Ià G* % z hde PrintI StatisticsIg Ø Æ START 54 22 G¥ Æ * % z hIä å ^ Ë f Ig test 1IØ Æ Save Õ° L ® ® æ { K ^ f Ë - ç ] è é ì BD FACSCanto II 55 5.7 Log Out 2 + 23 GD E hþ ê w " #IZ | } pI " # h-i- \ FACSDiva [ r ¥ 24 & + £ H z Õ° é w Î t File >> Quit ' File >> Log OutIà G¥ Æ * % z hd Quit / Ú è [ 5.8 3 25 Õ° Excel 4 5 6 * + , - 7 Excel® æ >> Microsoft Excel 26 Õ° ë Ià G ç ª h D:\BD Export\Statistic ì í w Í Î CSV 56 27 Z î ï - - Idñ 9 8 30 { R 9 8 ì í g BD FACSCanto II 57 +, +,- ' ÙÚ¾ . : / Û 0 (PMTs 1 2 3 X Ü 4-2 4 5 6 7 ; < (nm) ) : ; < (nm) Fluorescein, FITC 490 520 (Blue E) Phycoerythrin-R, PE 495 578 (Blue D) Peridinin-chlorophyll, 490 677 (Blue B) PerCP-Cy5.5 490 (Blue B) Cy-Chrome 495 670 (Blue B) PE-Texas Red 495 620 (B or D) PE-Cy5 495 670 (Blue B) PE-Cy7 490 781 (Blue A) Allophycocyanine 650 660 (Red C) APC-Cy7 650 781 (Red A) Ethidium Bromide 510 (+ds DNA) 595 (Blue D) Propidium Iodide 536 (+ds DNA) 623 (B or D) Acridine Orange 480 (+ DNA) 440-70 (+ RNA) 520 (Blue E) 650 (Blue B) Thiazole Orange 509 (+ RNA) 533 (Blue E) Propidium Iodide 536 623(B or D) YOPRO-1 480 515 (Blue E) Acridine Orange 480 520 (Blue E) 7-AAD 488 670 (Blue B) DiO-C6 (3) 485 510 (Blue E) Rhodamine 123 485 546 (Blue E) BCECF-AM 488 Ratio 520/620 SNARF1-AM 514 Ratio 587/640 Fluo4-AM Calcium Green-1 488 488 528 (Blue E) 530 (Blue E) Fura Red 488 660 (Blue B) H2O2 sensitive Dihydrorhodamine 123 DCFH-DA 505 505 534 (Blue E) 535 (Blue E) O2- radical sensitive Hydroethidine 505 600 (Blue D) Esterase sensitive Fluorescein -DA 495 525 (Blue E) 6l±DAÏ PerCP 1 ²³ 2 Viability h Ü ´ Mitochondrial h Ü ª ´ ª pH < µ ) 58 Optical Configurations +, á ò ? d nm % R TM Coherent Sapphire ÷ 3 @ AB mW % 488C % 20 FITC, PE, PE-Texas Red, PerCP, PerCP-Cy5.5, PE-Cy7, PI TM JDS Uniphase HeNe 633D % 17 APC, APC-Cy7 Point Source iFlex2000 405¶ % 30 Pacific Blue, AmCyan Ò 4 + , 5 6 7 488 Laser 8 9 3 4 5 : ; < = ( . 4-2-2 / 0 ) PMT LP Filter BP or LP Filters Fluorochromes Position nm% nm% Blue A: 735 780/60 PE-Cy7 Blue B: 655 670LP PerCP, PerCP-Cy5.5 633 Laser 405 Laser Blue C: 610 Blank filter N.A Blue D: 556 585/42 PE Blue E: 502 530/30 FITC Blue F: Blank filter 488/10 SSC Red A: 735 780/60 APC-Cy7 Red B: 685 Blank filter N.A Red C: Blank filter 660/20 APC Viiolet A: 502 510/50 AmCyan Violet B: Blank filter 450/50 Pacific blue BD FACSCanto II 59 + , 6 7 488 Laser 8 9 3 4 5 : ; < = ( . 5-3 / 0 ) PMT LP Filter BP or LP Filters Fluorochromes Position nm% nm% Blue A: 735 780/60 PE-Cy7 Blue B: 655 670LP PerCP, PerCP-Cy5.5 633 Laser Blue C: 610 616/23 PE-Texas Red Blue D: 556 585/42 PE Blue E: 502 530/30 FITC Blue F: Blank filter 488/10 SSC Red A: 735 780/60 APC-Cy7 Red B: 685 712/21 Alexa Fluor-700 Red C: Blank filter 660/20 APC 60