BD LSRII User manual

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LSR!II!Users!Guide!
Stanford!Shared!FACS!Facility!
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##$!%&'(!)*!+,-.!/(!/00!(1%*'!1.!,&-!23456!*.71-,.%*.(!
84/)!9,/(:!;0,7*':!/.<!90,'*<!(,*<!'=,*'!/-*!%/.</(,->?!-*'@1-/(,-!/.<!;,;;0*'!/-*!/7/10/)0*8!
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A.!B.'(-&%*.(!C,.D1;&-/(1,.!1'!0,9/(*<!,.!(=*!+*)'1(*!1.!(=*!
B.'(-&%*.(!E7*-71*+!/-*/!!
8F=*!9,.D1;&-/(1,.!01'('!(=*!0/'*-'!/.<!D10(*-'!D,-!*/9=!%/9=1.*8!
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Do!I!need!to!run!beads!when!I!collect!data!using!‘Diva!Digital’?!
G,-!*/9=!</(/!9,00*9(1,.:!>,&!.**<!(,!D1-'(!9,00*9(!</(/!,.!"HI!9/01)-/(1,.!)*/<'J!!F=1'!1'!(,!*.'&-*!(=/(!(=*!
1.'(-&%*.(!1'!-&..1.;!@-,@*-0>J!
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What!samples!do!I!need!to!bring?!
• A00!&'*-'!ALWAYS!.**<!(,!)-1.;!'1.;0*!'(/1.*<!9,%@*.'/(1,.!9,.(-,0'!
• BD!>,&!=/7*!/!I!9,0,-!@/.*0!>,&!'=,&0<!)-1.;K!!L!9,%@*.'/(1,.!9,.(-,0'!
o One$tube$stained$with$each$individual$antibody$$
o One$unstained$tube$
o No$PI$or$viability$dye$in$the$single$color$control$
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Don’t!have!enough!cells!for!compensation?!
M'*-!9/.!@&-9=/'*!9,%@*.'/(1,.!)*/<'!D-,%!%/.>!<1DD*-*.(!'&@@01*-'!
• *21,'91*.9*!M0(-/C,%@!2*/<'!N"566665O6!(for$panels$including$UV$and$Violet$dyes)!
• *21,'91*.9*!E.*C,%@!2*/<'!N"5""""5O6!(for$panels$that$do$NOT$include$any$UV$or$Violet$dyes)!
• 2P!C,%@2*/<'!QQ6LOQ!RA.(15S/(TU/%'(*-V!(for$most$anti-mouse$antibodies)!
• 2P!C,%@2*/<'!QQ6LOW!RA.(15X,&'*V!(for$most$anti-human$antibodies)!
• F=*-%,G1'=*-!A%1.*!S*/9(17*!C,%@*.'/(1,.!2*/<'!A"NY6L!(for$LIVE/DEAD$Fixable$stains)!
Starting!Up!the!System!
8A00,+!WN!%1.&(*'!D,-!(=*!0/'*-'!(,!+/-%!&@8!
"J F&-.!,.!(=*!C>(,%*(*-J!!
/J F=*!@,+*-!'+1(9=!1'!0,9/(*<!,.!(=*!0,+*-!-1;=(!9,-.*-!,.!(=*!C>(,%*(*-J!!
)J F=*!D0&1<!9,.(-,0!)&((,.!01;=('!/-*!GREEN!/.<!(=*!STANDBY!)&((,.!01;=(!1'!/%)*-J!
6J 33GG!9,%@&(*-'!/-*!0*D(!,.!/(!/00!(1%*'J!
/J BD!9,%@&(*-!1'!(&-.*<!,DD:!0,;!1.(,!Z1.<,+'!+1(=!M'*-![/%*K!!GAC3!M'*-!!!
1J F=*-*!1'!.,!@/''+,-<!!

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LSR-II!20L!Pump!System!Instructions!(Cytek)!
G0&1<!%/./;*-'!/-*!1.!&'*!,.!/00!,&-!43S!BB'J!M'1.;!(=*!@&%@!,DD*-'!(=*!D,00,+1.;!/<7/.(/;*'K!
• [,!.**<!(,!,@*.!/.<!D100!/!%*(/0!(/.\J!
• 3(/)101]1.;!,D!'=*/(=!D0,+!-/(*'!/00,+!&'*!,D!/!Y!&3*9!+1.<,+!*^(*.'1,.J!
To!use:!
"J F=*!@&%@!'>'(*%!(&-.'!,.!/&(,%/(19/00>!+1(=!(=*!43SJ!!
6J _*-1D>!(=/(!(=*!@&%@!1'!,.J!!F=*!43S!+100!.,(!+,-\!@-,@*-0>!,(=*-+1'*J!
Its$switch$is$located$on$the$blue$box$labeled$“Cytek”$under$the$LSR$with$the$black$lever$switch.$
WJ C=*9\!(=*!'=*/(=!(/.\J!G100!(=*!'=*/(=!(/.\!1D!(=*-*!1'!.,(!*.,&;=!D,-!>,&-!-&.J!!
OJ C=*9\!(=*!+/'(*!(/.\J!$%@(>!(=*!+/'(*!(/.\!1D!D&00J!
QJ #0*/'*!*%/10!facs[email protected]!+1(=!/.>!@-,)0*%'!,-!`&*'(1,.'!
Changing!the!tanks:!
"J F=*!9&)*!1'!,.!/!'9/0*:!',!>,&!9/.!9=*9\!(=*!+*1;=(!,D!(=*!9&)*!a/-*!(=*-*!;&1<*01.*'!D,-!+=/(!(=*!
-*/<,&(!'=,&0<!)*b!!B.'(-&%*.(!&'*!1'!c"4T=!,-!6J60)'dJ!!
6J BD!(=*!'=*/(=!9&)*!1'!*%@(>!-*@0/9*!1(!+1(=!/!.*+!,.*!a1(!1'!=*/7>!',!01D(!9/-*D&00>d![*+!9&)*'!/-*!
0,9/(*<!1.!*1(=*-!(=*!=/00!,-!(=*!-*/-!,D!(=*!-,,%J!!
Note:$The$cube$closure$just$screws$on$–$there$is$no$pressurization$of$the$cube.$Be$careful$not$to$kink$the$
tubing.$$Turn$the$outer$ring$only$so$you$do$not$stress$the$wires$for$the$sensors.$
WJ $%@(>!+/'(*!1.!'1.\!1.!=/00+/>!+=*.!D&00:!&'1.;!(=*!@-,(*9(17*!D/9*!'=1*0<:!;0,7*'!/.<!0/)!9,/(J!!
Remember:$Add$bleach$to$line$marked$on$carboy.!
OJ A0/-%!'*.',-'!/-*!9,..*9(*<!(,!(=*!9&)*!/.<!(=*!+/'(*:!1.<19/(1.;!+=*.!(=*>!.**<!/((*.(1,.!1D!>,&!
D,-;*(J!!F=*!43S!+,.V(!-&.!'/%@0*'!@-,@*-0>!1D!(=*!@&%@!1'!(&-.*<!,DDJ!!!
The$pump$should$NEVER$be$turned$off$manually!!$
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Diva!Software!Instructions!
"J 4,;!1.(,!(=*!P17/!',D(+/-*!&'1.;!2PGAC3P17/!',D(+/-*!19,.!,.!(=*!<*'\(,@!
Username$is$Sunet$ID/$password$starts$with$a$capital$letter$
6J Z/(9=!D,-!(=*!eB.'(-&%*.(!9,..*9(*<f!'(/(&'!1.!),((,%!,D!(=*!C>(,%*(*-! +1.<,+!
WJ BD!C3F!%1'%/(9=!<1/0,;!),^!/@@*/-'!'*0*9(!&'*!CST!settings !
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Setting!Up!Your!Experiment!
"J C-*/(*!/.!$^@*-1%*.(K!3*0*9(!$^@*-1%*.(®[*+!$^@*-1%*.(®3*0*9(!(=*!33GG!(/)®9=,,'*!(=*!
.*+*'(!</(*!/7/10/)0*®'*0*9(!*^@*-1%*.(!C019\®!Eg!
6J F,!S*./%*!55!-1;=(59019\!®S*./%*!!!!!!!!Use$descriptive$name$and$date!
WJ $<1(!%*.&®M'*-!#-*D*-*.9*'®3*0*9(!<*'1-*<!<*D/&0('J!!$
You$can$deselect$save$analysis$and$load$data$after$acquisition$to$stop$instrument$from$reloading$data$after$
acquisition.$$This$helps$if$you$are$recording$large$data$files.$
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Calibration!!
"J S*%,7*!(=*!+/(*-!(&)*!D-,%!(=*!3B#!
6J #&'=!(=*!RUN!)&((,.!,.!D0&1<19'!@/.*0!/.<!.,(*!(=*!<-,@!,D!'/01.*!)/9\!D0&'=1.;!D-,%!(=*!3B#!
WJ #&'=!(=*!LO!)&((,.!,.!C>(,%*(*-!@/.*0!
OJ #0/9*!(=*!'(/.</-<!"HI!9/01)-/(1,.!)*/<'!a0,9/(*<!1.!-/9\!,-!D-**]*-d!,.!(=*!3B#!
QJ h,!(,!R2*/<'!3@*91%*.!a'>-1.;*!19,.dV!!
YJ C4BCg!,.!RiV!'1;.!
IJ C019\!,.!R"HI!2*/<'V!(&)*!aF=*!;-**.!/--,+
à
!'=,&0<!)*!@,1.(1.;!/(!(=*!
(&)*d!This$makes$acquisition$controls$active!!!!
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LJ B.!Acquisition!Dashboard!C4BCg!àAcquire!Data!
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HJ 3*(!$7*.('!(,!P1'@0/>!(,!QN5"NN!D,-!9/01)-/(1,.!
"NJ 3*(!D0,+!-/(*!(,!LO!
""J AD(*-!)*/<'!/@@*/-!,.!@0,(':!&'*!D1.*!(&.*!a)0/9\!\.,)d!(,!/<j&'(!D0,+!-/(*!(,!/@@-,^J!6NN!)*/<'T'*9!
"6J C=*9\!(=/(!(=*!e<*0/>!+1.<,+!*^(*.'1,.f!;0,)/0!+,-\!'=**(!1'!'*0*9(*<J!!
"WJ G,00,+!(=*!,.5'9-**.!1.'(-&9(1,.'!/.<!9=*9\!(=*!1.'(-&%*.(!0/'*-!<*0/>':!/.<!/-*/!'9/01.;J!
/J C,.D1-%!(=*!G,-+/-<!'9/((*-!(=-*'=,0<!1'!'*(!(,!QNk!,D!(=*!D,-+/-<!'9/((*-!'1;./0J!
)J C=*9\!(=*!0/'*-!<*0/>'!)>!90,'1.;!(=*!+1.<,+!*^(*.'1,.!(,!NJ!!
9J F=*!/-*/!'1;./0!D,-!(=*!h-**.:!S*<!/.<!_1,0*(!0/'*-'!'=,&0<!<-,@!.,!%,-*!(=/.!QNkJ!!
(You$can$use$the$height$value$for$a$quick$reference$to$100$%$signal.)$
<J BD!(=*!<*0/>'!/-*!1.9,--*9(!D1-'(!9=*9\!(=*!C3F!7/0&*'J!M'*!(=*!0/(*'(!@*-D,-%/.9*!'=,-(9&(!,.!(=*!
<*'\(,@!D,-!(=*!%,'(!-*9*.(!C3F!-&.J!
*J E@*.!(=*!+1.<,+!*^(*.'1,.!)/9\!(,!YJNN!
DJ C=*9\!(=/(!A-*/!39/01.;!7/0&*'!/-*!9,--*9(J!!A-*/!'=,&0<!)*!;-*/(*-!(=/.!,-!*`&/0!(,!(=*!=*1;=(!
+1(=1.!"NkJ!BD!-*0/(1,.'=1@!1'!,DD:!%/\*!/<j&'(%*.('!1.!(=*!/-*/!'9/01.;!'*9(1,.!1.!(=*!0/'*-!(/)J!!
"OJ 3(,@!A9`&1-1.;J!
Acquisition$must$be$stopped$in$order$to$change$the$worksheets$
"QJ 3+1(9=!(,!(=*!e'(/.</-<'f!;0,)/0!+,-\'=**(J!
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"YJ A<j&'(!(=*!#XF!7,0(/;*'!(cytometer$window$ à $parameters$tab)!',!(=*!%*/'&-*<!7/0&*'!/-*!/(!,-!
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"IJ #-*''!RECORD!DATA!!1.!(&)*!0/)*0*<!R"HI!)*/<'V!
Experiment!Definition!
"J !3*0*9(!!(=*!.*^(!'@*91%*.!1.!(=*!*^@*-1%*.(!Specimen!C019\!(=*!R+V!(,!'**!
Tube_001!
6J U1;=01;=(!Tube_001!1.!.*+!'@*91%*.!/-*/!à9019\!,.!/--,+!,.!0*D(!
WJ B.!(=*!C>(,%*(*-!@/.*:!<*0*(*!@/-/%*(*-'!>,&!+100!.,(!)*!&'1.;J!!
/J B.!#/-/%*(*-'!(/):!9019\!,.!'%/00!)&((,.!(,!0*D(!,D!@/-/%*(*-!./%*J!!
)J C019\!<*0*(*!)&((,.$$(use$shift$key$and$highlight$for$multiple$deletions)!

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9J S*@*/(!D,-!*/9=!@/-/%*(*-!>,&l-*!.,(!&'1.;!
<J P,!.,(!<*0*(*!#/-/%*(*-'!(=/(!%/>!)*!,D!1.(*-*'(J!!!
*J BD!>,&!/<<!)/9\!/!<*0*(*<!@/-/%*(*-!(,!(=*!*^@*-1%*.(!1(!+100!9,%*!
1.!+1(=!<1DD*-*.(!7,0(/;*':!.,(!'@*91D1*<!#XF!'*((1.;'!)>!D/9101(>J!
F=1'!%/>!9/&'*!@-,)0*%'!D,-!-*',0&(1,.J!C=*9\!9>(,%*(*-!
'*((1.;'!D,-!(=*!"HI!)*/<!(&)*!(,!'**!9,--*9(!#XF!7,0(/;*!(,!
/''1;.!+=*.!/<<1.;!)/9\!/!@/-/%*(*-J!
OJ g**@!D,-+/-<!/.<!'1<*!'9/((*-!=*1;=(!/.<!+1<(=!'*0*9(*<!
QJ F=*!</(/!%/>!)*!71*+*<!9,%@*.'/(*<!,-!.,(:!)&(!(=*!&.9,%@*.'/(*<!
</(/!1'!+-1((*.!(,!(=*!</(/)/'*!/0,.;!+1(=!(=*!%/(-1^!',!(=/(!,(=*-!
9,%@*.'/(1,.'!%/>!)*!&'*<!0/(*-!1.!G0,+m,J!
Note:$$For$each$tube$collected$in$Diva$software,$uncompensated$data$is$
recorded$and$the$compensation$matrix$is$applied$as$a$transform,$it$does$not$
affect$your$data.$
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A00!'/%@0*'!%&'(!)*!D10(*-*<!D,-!',-(*-'J!F=*!/./0>'1'!1.'(-&%*.('!=/7*!.>0,.!
%*'=!'`&/-*'!,.!(=*!<*'\(,@!1D!>,&!.**<!(=*%!
Compensation!
• C,--*9('!D,-!(=*!*%1''1,.!,D!,.*!D0&,-!1.(,!(=*!<*(*9(,-!&'*<!(,!%*/'&-*!/.,(=*-J!
• G0,+!C>(,%*(*-!&'*!D10(*-!'*('!(=/(!/-*!,@(1%1]*<!D,-!7*->!'@*91D19!+/7*0*.;(='!,D!01;=(J!
• P>*TG0&,-,9=-,%*!*%1''1,.'!,D(*.!'@/.!D-,%!,.*!D10(*-!1.(,!/.,(=*-J!
• CEX#$[3AFBE[!B3!_$Sn!BX#ESFA[F!5!!+1(=,&(!1(:!,&-!</(/!9/..,(!)*!1.(*-@-*(*<!@-,@*-0>J!
• F,!9/09&0/(*!9,%@*.'/(1,.:!*7*->!*^@*-1%*.(!.**<'!/.!&.'(/1.*<!9,.(-,0!/'!+*00!/'!*/9=!
D0&,-,9=-,%*!'1.;&0/-0>J!
• A.(1),<>!9/@(&-*!)*/<'!9/.!)*!'&)'(1(&(*<!D,-!9*00!9,.(-,0'J!
"J C-*/(*!C,%@*.'/(1,.!C,.(-,0'J!3*0*9(!$^@*-1%*.(!à!
C,%@*.'/(1,.!3*(&@!à!C-*/(*!C,%@*.'/(1,.!C,.(-,0'J!!
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6J B.!(=*!C-*/(*!C,%@*.'/(1,.!C,.(-,0'!
+1.<,+:!*.'&-*!(=/(!/00!,D!>,&-!9,0,-'!/-*!
0/)*0*<!eh*.*-19fJ!F=*.!'*0*9(!eEgfJ!Do$
not$compare$negative$cells$to$positive$
beads$or$vice$versa.$!
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Q!
WJ P17/!+100!9-*/(*!/!.*+!'@*91%*.!9/00*<!eC,%@*.'/(1,.!C,.(-,0'fJ!$^@/.<!(=*!'@*91%*.!/.<!/9(17/(*!
(=*!D1-'(!(&)*J!!
OJ 3*(!$7*.('!(,!<1'@0/>!(,!QNNNJ!
QJ S&.!&.'(/1.*<!9*00'!,-!)*/<'!D1-'(J!!
YJ A<j&'(!(=*!G3C!/.<!33C!7,0(/;*'!(,!@0/9*!(=*!@,@&0/(1,.!,D!1.(*-*'(!,.!'9/0*J!
IJ A<j&'(!(=*!G3C!(=-*'=,0<!7/0&*!(,!*^90&<*!%,'(!,D!(=*!<*)-1'!+1(=,&(!*^90&<1.;!(=*!@,@&0/(1,.!,D!
1.(*-*'(J!!
Setting$it$to$50%$of$the$smallest$cell$of$interest$works$well.!
LJ X,7*!(=*!#"!;/(*!,.!(=*!G3CT33C!@0,(!(,!1.90&<*!>,&-!9*00'!,D!1.(*-*'(J!!
HJ #&(!>,&-!D&00>!'(/1.*<!'/%@0*!,.J!X/\*!'&-*!(=*!@,'1(17*'!/-*!,.!'9/0*J!BD!>,&-!@,'1(17*'!/-*!/),7*!"NQ!
>,&!+100!.**<!(,!<*9-*/'*!(=*!7,0(/;*J!
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*Remember,$if$you$significantly$lower$the$PMT$voltage$below$the$baseline$settings$in$order$to$bring$the$
positive$population$on$scale,$then$the$dim$populations$might$not$be$easily$resolved$from$the$negative$
populations$for$that$parameter.$It$is$acceptable$to$have$the$negative$peak$shift$above$the$first$log$decade$in$
some$detectors.$
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"NJ C=*9\!*/9=!'1.;0*!9,0,-!1.!(=*!&.'(/1.*<!9*00!(&)*!/-*/!',!+*!9/.!<*D1.*!(=/(!/00!*7*.('!/-*!,.!'9/0*J!
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M'*!/.!1.(*-7/0!;/(*!(,!9-*/(*!/!#W!;/(*!/-,&.<!>,&-!.*;/(17*!@*/\J!!
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"6J 3*0*9(!$^@*-1%*.(!à!C,%@*.'/(1,.!3*(&@!à!C/09&0/(*!C,%@*.'/(1,.J!Z=*.!(=*!31.;0*!3(/1.*<!3*(&@!
),^!/@@*/-':!'*0*9(!eA@@0>!E.0>f!(,!/@@0>!(=*!9/09&0/(*<!7/0&*'!(,!>,&-!*^@*-1%*.(J!
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Y!
*Error$messages$warn$you$of$compensation$values$that$are$>100%.$This$can$indicate$one$of$two$
things,$the$voltage$settings$used$are$such$that$one$of$your$dyes$is$brighter$in$an$off-channel$than$it$is$
in$the$channel$selected$and$a$channel$receiving$less$total$photons$has$been$over$amplified,$or$that$the$
color$combination$at$the$settings$you$have$chosen$is$not$optimal.$$If$you$are$still$having$problems,$it$
may$indicate$that$you$do$not$have$a$good$color$combination.*$
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"WJ S*(&-.!(,!>,&-!,-1;1./0!h0,)/0!Z,-\'=**(!)>!9019\1.;!,.!(=*!h0,)/0!Z,-\'=**(!19,.!1.!(=*!Z,-\'=**(!
Z1.<,+J!
Running!Samples!
"J C-*/(*!/!G3C5A!)>!33C5A!@0,(!,.!(=*!;0,)/0!+,-\'=**(!/.<!9-*/(*!/!@,0>;,.!;/(*!(,!'*0*9(!>,&-!9*00'J!!
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6J C-*/(*!/!@,@&0/(1,.!=1*-/-9=>!)>!-1;=(59019\1.;!,.!/.>!<,(!@0,(!/.<!
'*0*9(1.;!e3=,+!#,@&0/(1,.!U1*-/-9=>fJ!C-*/(*!,(=*-!<,(!@0,('!,-!
=1'(,;-/%'!/'!.**<*<!D,-!>,&-!*^@*-1%*.(J!!
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WJ X/\*!'&-*!(=*!%/9=1.*!1'!1.!RUNJ!
OJ 4,/<!>,&-!D1-'(!'/%@0*!(&)*!,.(,!(=*!1.'(-&%*.(!/.<!9019\!eACQUIRE!
DATAfJ!!
QJ A<j&'(!>,&-!GC3!/.<!33C!7,0(/;*'!1D!.**<*<J!!
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9019\!eRECORD!DATAfJ!!
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/./0>'1':!*^@,-(!(*%@0/(*:!-*./%*:!/.<!*^@,-(!(,!;*.*-/0J!

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Clean-up!
"J S&.!BLEACH!,.!HIGH!D,-!Q!%1.&(*'!!
6J S&.!WATER!,.!HIGH!D,-!Q!%1.&(*'!
WJ 4*/7*!(=*!%/9=1.*!1.!STANDBY!/.<!LOW!+1(=!/!(&)*!,D!WATER!,.!(=*!3B#J!
IF!you!ran!BSL-2!samples!follow!the!additional!cleaning!steps!below:!
OJ 3@-/>!9/7191<*!,.!/00!'&-D/9*'J!C,.(/9(!(1%*!D,-!9/7191<*!1'!Q!%1.&(*'!&.0*''!>,&-!A#2!-*`&1-*'!/!
0,.;*-!@*-1,<J!!Please$let$staff$know$if$different$contact$times$are$needed.!
QJ G1005,&(!(=*!)1,'/D*(>!901@),/-<!.*^(!(,!(=*!%/9=1.*J!41'(!(=*!)1,=/]/-<:!&'*-:!0/)!/.<!90*/.!&@!
@-,9*<&-*J!!
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+1(=!@/@*-!(,+*0'?!<1'@,'*!,D!(=*!@/@*-!(,+*0'!1.!(=*!)1,=/]/-<!+/'(*!9,.(/1.*-J!!
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Rules!
Data!Rules!
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WJ n,&!XM3F!*^@,-(!>,&-!*^@*-1%*.(J!!
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QJ n,&!XM3F!D,00,+!(=*!90*/.1.;!@-,9*<&-*J!
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Notes!on!Data!
• C=*9\!1.!>,&-!</(/!(,!@-,(*9(!1(!
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• C&--*.(0>!+*!/-*!-*9,%%*.<1.;!G0,+m,!7*-'1,.!"NJ6!
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• n,&!+100!)*!@-,%@(*<!D,-!M'*-./%*K!RD0,+j,V!#/''+,-<K!RW"O"QHVJ!!