BD FACSJAZZ User manual

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lasted!updated!by!MW!on!11/10/2017!
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BD#FACSJazz#Users#Guide#
Satchmo#
Stanford#Shared#FACS#Facility#
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PPE!must!be!worn!at!all!times!in!our!BSL-2!environment!
*Lab!coat,!gloves,!and!closed!toed!shoes!are!mandatory;!respirator!and!goggles!are!available*!
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An!Instrument!Configuration!is!located!on!the!website!in!the!
Instrument!Overview!area!!
*The!configuration!lists!the!lasers!and!filters!for!each!machine*!
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All#samples#must#be#in#POLYPROPYLENE#round#bottom#tubes.#
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What#samples#do#I#need#to#bring?#
• All!users!ALWAYS!need!to!bring!single!stained!compensation!controls!
• If!you!have!a!4!color!panel!you!should!bring:!!5!compensation!controls!
o One$tube$stained$with$each$individual$antibody$$
o One$unstained$tube$
o No$PI$or$viability$dye$in$the$single$color$controls$
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Don’t#have#enough#cells#for#compensation?#
User!can!purchase!compensation!beads!from!many!different!suppliers!
• eBioscience!UltraComp!Beads!01-2222-42!(for$panels$including$UV$and$Violet$dyes)!
• eBioscience!OneComp!Beads!01-1111-42!(for$panels$that$do$NOT$include$any$UV$or$Violet$dyes)!
• BD!CompBeads!552845!‘Anti-Rat/Hamster’!!
• BD!CompBeads!552843!‘Anti-Mouse’!
• ThermoFisher!Amine!Reactive!Compensation!Beads!A10628!(for$LIVE/DEAD$Fixable$stains)!
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lasted!updated!by!MW!on!11/10/2017!
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Before#you#begin:#
1. Make!sure!the!plates!are!clean.!You!may!choose!to!take!them!out!and!clean!them.!
2. Close!the!FACSSortware.!
3. Close!the!Pressure!Controls!choosing!Just#Quit!
4. Log!out!of!Windows!(please!make!sure!you!sign!out)!
5. Log!in!to!Windows!with!Your!SUNet!ID!
6. Open!FACS!Sortware!and!push!CONNECT!
Stream#Start#up#
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*Note:$there$is$an$image$of$the$knobs$on$the$at$the$end$of$this$section$to$guide$you$in$which$knob$is$which.*$
1. Restore!workspace—SSFF!template!“most!recent!date”!
2. In!the!sort!settings!increase!the!piezo!amplitude!to!between!3!and!6.!
3. Press!the!ILLUM!button.!
4. Look!at!the!third!window!of!the!stream!camera.!
5. Use!the!left/right!and!the!pitch!black!knobs!to!adjust!the!stream!so!that!it!is!falls!into!the!aspirator!
and!that!it!is!bright!and!tight.!
*like$the$image$below*$
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6. Adjust!the!stream!so!that!the!stream!is!centered!over!the!pinholes!and!in!focus.!Using!the!black!
left/right!and!the!focus!adjustment!knobs.!
*like$the$image$below*$
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7. You!may!have!to!repeat!5!&!6!to!get!the!adjustments!correct.!$
8. Using!the!vertical!adjustment!knob,!adjust!the!nozzle!so!that!it!is!sitting!at!the!top!edge!of!the!panel.!
9. Adjust!the!piezo!amplitude!such!that!there!is!a!proper!gap!and!break!of!point.!This!can!be!monitored!
in!the!second!camera!view!panel.!The!stream!should!look!similar!to!this.!
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lasted!updated!by!MW!on!11/10/2017!
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10. Click!the!PLATES#button!to!turn!on!the!plates.!
11. Close!the!shutter!so!that!the!plates!will!be!activated.!
12. Click!TEST#STREAM!in!the!sort!settings!window.!(You$
should$see$a$deflected$drop$to$the$left$of$your$stream$in$the$
3rd$window$of$the$streams.)!
13. Click!FLASH#CHARGE!and!adjust!the!piezo!amplitude!till!
the!deflected!drop!stays!out!to!the!left.!
14. Click!SHORT#FLASH!and!adjust!the!piezo!amplitude!till!the!
deflected!drop!stays!out!to!the!left.!
*like$the$image$below*$
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**Please$note$the$ILLUM$button$and$the%PLATES%button**$
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**Please$note$the$diagram$of$the$nozzle$assembly$with$the$labels$of$each$knob.**$
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Step!8à!
Step!5&6à!
Step!5&6â!
Step!5&6â!

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lasted!updated!by!MW!on!11/10/2017!
4!
Bead#Set#up#
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197#Beads#
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1. Close!the!laser!shutter,!activate!the!shutter!and!make!sure!the!lasers!are!on.!
2. Make!sure!you!have!already!restored!the!proper!workspace.!!
3. Vortex!and!load!beads!onto!sample!port!and!press!SAMPLE#and!press!BOOST!to!get!
them!sample!running.!
4. Press!ACQUIRE!in!the!acquisition!bar.!
5. Adjust!the!event!rate!to!about!200events/second!by!adjusting!the!offset!to!about!.25PSI!
6. In!the!recording!settings!adjust!the!events!to!display!to!200.!
7. Align!the!beads!into!their!target!boxes.!
a. Using!the!color!adjustments!knobs!
b. Using!the!focus!adjustment!knob!
8. Press!SAMPLE#to!stop!the!beads!from!running.!
And!STOP#ACQUIRING!in!the!software.!
9. Remove!the!beads!from!the!sample!port.!
10. Press!BACKFLUSH,!to!remove!the!beads!from!the!
sample!line!
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lasted!updated!by!MW!on!11/10/2017!
5!
Accudrop#Beads#
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1. Adjust!the!piezo!amplitude!such!that!there!is!a!proper!gap!and!break!of!
point.!This!can!be!monitored!in!the!third!panel.!The!stream!should!look!
similar!to!this.!
2. Click!on!the!PLATES#button!to!turn!on!the!plates.!
3. Click!on!TEST#STREAM!in!the!sort!settings!window.$(You$should$see$a$2nd$
drop$to$the$left$of$your$stream$in$the$3rd$window$of$the$streams.)!
4. Click!FLASH#CHARGE!and!adjust!the!piezo!amplitude!till!the!second!drop!
stays!out!to!the!left.$!
5. Click!SHORT#FLASH!and!adjust!the!piezo!amplitude!till!the!deflected!drop!stays!out!to!the!left.!
*like$the$image$below*$
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6. Vortex!and!load!beads!on!to!sample!port!and!press!SAMPLE#and!press!BOOST!to!get!them!sample!
running.!
7. Press!ACQUIRE!in!the!acquisition!bar.!
8. Adjust!the!event!rate!to!between!1000events/second!and!1500events/second!by!adjusting!the!offset!
to!about!1.5!PSI!
9. In!the!Sort!Layout!window,!make!sure!the!sort!device!
says!Accudrop!Setup,!and!it!is!sorting!P1!to!the!left!
10. Make!sure!the!sort!mode!is!1.0#drop#pure.!
11. Click!ACCUDROP!in!the!Pressure!Console!to!turn!on!
the!accudrop!filter.!
12. Click!START!in!the!sort!layout!window.$!
13. Adjust!the!drop!delay!value!until!you!only!see!the!left!
stream!visible!and!the!center!stream!disappears.!!
*Should$look$like$the$images$below*$
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14. Click!ACCUDROP!in!the!control!window!to!turn!off!the!accudrop!
filter.!
15. Click!STOP!to!stop!the!sort.!!
16. Press!SAMPLE#to!stop!the!beads!from!running.!
17. Remove!the!beads!from!the!sample!port.!
18. Press!BACKFLUSH,!to!remove!the!beads!from!the!sample!line.$!
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