BD LSR II User Manual

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Page 1

LSR!II!Users!Guide!

Stanford!Shared!FACS!Facility!

##$!%&'(!)*!+,-.!/(!/00!(1%*'!1.!,&-!23456!*.71-,.%*.(!

84/)!9,/(:!;0,7*':!/.<!90,'*<!(,*<!'=,*'!/-*!%/.</(,->?!-*'@1-/(,-!/.<!;,;;0*'!/-*!/7/10/)0*8!

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!

! 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!

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$control$

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!!

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6J 33GG!9,%@&(*-'!/-*!0*D(!,.!/(!/00!(1%*'J!

/J BD!9,%@&(*-!1'!(&-.*<!,DD:!0,;!1.(,!Z1.<,+'!+1(=!M'*-![/%*K!!GAC3!M'*-!!!

1J F=*-*!1'!.,!@/''+,-<!!

Page 2

LSR-II!20L!Pump!System!Instr u ctio n s!(Cy te k)!

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:!

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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![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.!

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D,-;*(J!!F=*!43S!+,.V(!-&.!'/%@0*'!@-,@*-0>!1D!(=*!@&%@!1'!(&-.*<!,DDJ!!!

The$pump$should$NEVER$be$turned$off$manually!!$

! !

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 !

! ! ! ! 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!!

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(&)*d!This$makes$acquisition$controls$active!!!!

LJ B.!Acquisition!Dashboard!C4BCg!àAcquire!Data!

! ! ! ! ! !

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!

"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)!

Page 4

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.$ $

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!!

! ! ! ! ! ! ! ! ! 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$versa.$!

! !

Page 5

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!

*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$detectors.$

"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!!

! ! ! ! ! ! ! ! ! "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!

! ! ! ! ! ! ! ! ! ! !

Page 6

*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.*$

"WJ S*(&-.!(,!>,&-!,-1;1./0!h0,)/0!Z,-\'=**(!)>!9019\1.;!,.!(=*!h0,)/0!Z,-\'=**(!19,.!1.!(=*!Z,-\'=**(!

Z1.<,+J!

Running!Samples!

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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!! YJ 3*0*9(!(=*!.&%)*-!,D!*7*.('!(,!-*9,-<!1.!(=*!3(,@@1.;!h/(*!'*((1.;'!,.!(=*!A9`&1'1(1,.!P/'=),/-<!/.<!

9019\!eRECORD!DATAfJ!!

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/./0>'1':!*^@,-(!(*%@0/(*:!-*./%*:!/.<!*^@,-(!(,!;*.*-/0J!

Page 7

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:!

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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!!

"#$%&'!()*+!,#-#!./012!345!67!8957!8: ;15< != >74 5: ? @!/9!2 4A 7 !/1B 7 C!

!" #$%&'()*(+,-(*./-()0(1)23(-45-3%/-*+"( 6" 7-$-&+(845)3+à845-3%/-*+( 9" :.'-(;23-(1)23(<.+.(%;(=-%*>(-45)3+-<(+)(=DEFG?9;/( ?" #$%&'()H( @" A*(+,-(B-;'+)5C(&$%&'()*(I#="!,4/4!=JF=H%&( D" E,-(.553)53%.+-(-45-3%/-*+(;,)2$<(=-(<%;5$.1-<C(=2+(%0(*)+(;-$-&+(%+"(( F" #$%&'()H( G" H%*<(1)23(*./-(.*<(#$%&'()H(

(

Check!the!Schedule—Is!someone!running!less!than!2!hours!after!you!today?!

BD!Yes:!0*/7*!(=*!%/9=1.*!/'!1':!1.!STANDBYJ! BD!No:!D,00,+!(=*!shutdown!1.'(-&9(1,.'J!

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Rules!

Data!Rules!

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Running!the!Machine!Rules!

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Notes!on!Data!

• C=*9\!1.!>,&-!</(/!(,!@-,(*9(!1(!

• P/(/!1'!<*0*(*<!%,.(=0>J!BD!1(!1'!.,(!9=*9\*<!1.!1(!1'!;,.*J!

• M32!/.<!@*-',./0!)/9\!&@!<*719*'!/-*!.,(!/00,+*<!,.!,&-!%/9=1.*'J!

• P1'9'!%/>!)*!)&-.*<!+=*.!/0(*-./(17*'!/-*!.**<*<J!

• A!Jj,!D10*!D,-!XAC!a0*;/9>!G0,+j,!HJIQ!/.<!,0<*-d!/.<!/!J+'@!a7*-'1,.!"N!),(=!X/9!/.<!#CdJ!Z100!)*!'*.(!

(,!>,&!(=-,&;=!(=*!</(/!/-9=17*!,.9*!>,&-!</(/!1'!9=*9\*<!1.!!

• C&--*.(0>!+*!/-*!-*9,%%*.<1.;!G0,+m,!7*-'1,.!"NJ6!

• 3(/-(!G0,+m,!/.<!D-,%!(=*!G10*X*.&!'*0*9(!E@*.!/.<!(=*.!,@*.!>,&-!J+'@!D10*J!!!

• n,&!+100!)*!@-,%@(*<!D,-!M'*-./%*K!RD0,+j,V!#/''+,-<K!RW"O"QHVJ!!