FluoProbes

FT-78932
Fluo-3,

Ca
2+
indicators
Product Information
Product name
Cat.number
MW
(gmol
-1
)

exc
\
em
max.
Free Ca
2+
(nm)

exc
\
em
max.
High Ca
2+
(nm)
mol. abs.
(M
-1
cm
-1
)
Kd
(nM)
Soluble
in
Fluo-! "M ester
FP-78932A
1129.8 !3 # $ea% ! # 2&
(a)
'M()
Fluo-! "M ester FPgra*e
FP-+12,A
1129.8 !3 # $ea% ! # 2&
(a)
'M()
Fluo-! "M ester
1 mM -n *r. 'M()
FP-M2!3&A
1129.8 !3 # $ea% ! # 2&
(a)
'M()
Fluo-! #$H4 salt
FP-37!2!A
8,.7 !3 # $ea% ! # 2& 1!! !!! 39!
(c)
/ater
012&
Fluo-! K salt
FP-!3&&91
9&!.! !3 # $ea% ! # 2& 1!! !!! 39!
/ater
012&
Fluo-! $a salt
FP-33!21A
879., !3 # $ea% ! # 2& 1!! !!! 39!
/ater
012&
(a) a4ter 5.*rol.6-6
(b) exc\em (7t)ac8no Mg29) : 3&&nm # ,7nm
(c) +e1orte* ;alue6 ;ar. 4rom 32 to ,& nm. <*=32nm *eterm-ne* at 22>C -n 1!! mM <Cl8 1! mM8 M)P(8 12 7.28 ! to 1! mM
Ca7?TA.
Storage% &ndicator salts can be 6tore* *e6-cate* an* 1rotecte* 4rom l-g5t at room tem1erature8
9,>C or @2!>C 01 .ear.
"M esters can be 6tore* *e6-cate* an* 1rotecte* 4rom l-g5t at @2!>C 0 & mont56.
Technical and Scientific Information
Fluo-
Fluo-3 an* t5e-r *er-;at-;e6 all ex5-b-t large 4luore6cence -nten6-t. -ncrea6e6 on b-n*-ng Ca
29
. Anl-%e -n*-cator6
4ura-2 an* -n*o-18 t5ere -6 no 61ectral 65-4t. T5e. are non-4luore6cent unt-l 5.*rol.Be* -ntracellularl. or -n t5e
1re6ence o4 Ca
29
. T5ere lo$er b-n*-ng a44-n-t. allo$6 mea6urement o4 5-g5er 1ea%6 o4 Ca
29
tran6-ent6 t5an $-t5
Fura 2.
Fluo-3 5a6 an ab6or1t-on 61ectrum com1at-ble $-t5 exc-tat-on at ,88 nm b. argon--on la6er 6ource68 an* a 01!!
4ol* 4luore6cence -nten6-t. -ncrea6e -n re61on6e to Ca
29
b-n*-ng. T5e6e 1ro1ert-e6 are ta%en to goo* account -n
m-cro6co1. an* Flo$ c.tometr..
Fluo-3 5a6 a 4luore6cence Cuantum .-el* (DE) o4 !.1 (Falue *eterm-ne* at 22>C -n 1!! mM <Cl8 1! mM8
M)P(8 12 7.2 conta-n-ng 39.8 GM 4ree Ca
29
).
"''lications%
M-cro6co1. -mag-ng 4or t5e 6tu*. o4 t5e 61at-al *.nam-c6 o4 man. elementar. 1roce66e6 -n Ca
29
6-gnal-ng.
Flo$ c.tometr. anal.6-6 4or ex1er-ment6 -n;ol;-ng 15otoact-;at-on o4 Hcage*I c5elator68 6econ*
me66enger6 an* neurotran6m-tter6
Cell-ba6e* a66a.68 e61ec-all. 4or te6t-ng xenob-ot-c6 e44ect on -ntracellular Ca
29
8 u1 to m-cro1late6
a66a.6 4or 15armaceut-cal 2-g5 T5oug51ut (creen-ng.
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FluoProbes

FT-78932
S'ectra o( Fluo-

Ab6or1t-on an* 4luore6cence em-66-on 61ectra o4 Ca
29
-6aturate*
Fluo-3 (AM 5.*rol.6e*) -n 12 7.2 bu44er.
")ailable (orms o( Ca
2+
indicators
T5e6e calc-um -n*-cator6 are a;a-lable a6 Acetox.met5.l e6ter (AM e6ter) an* 6alt6 4orm.
"M ester are membrane-1ermeant an* t5u6 -ncrea6e6 greatl. cell loa*-ng t5at can be 1er4orme* b. 6-m1le
-ncubat-on o4 t5e cell6 or t-66ue 1re1arat-on -n a bu44er conta-n-ng t5e AM e6ter. Pluron-c
K
F-1278 a m-l* non-
-on-c *etergent8 can 4ac-l-tate AM e6ter6 loa*-ng. T5e AM e6ter6 t5em6el;e6 *o not b-n* to Ca29. 2o$e;er8
once t5e. 5a;e entere* t5e cell68 t5e. are ra1-*l. 5.*rol.Be* b. -ntracellular e6tera6e6 -nto t5e 1arent Ca29
com1oun*68 t5u6 becom-ng 4ull. 4luore6cent u1on b-n*-ng to Ca29.
Salts (orm are membrane--m1ermeant8 but can be loa*e* -nto cell6 ;-a m-cro-nLect-on or 6cra1e loa*-ng.
Instruction for use
Handling and Storage
&ndicator salts = 6toc% 6olut-on6 o4 t5e 6alt6 ma. be 1re1are* -n *-6t-lle* $ater or aCueou6 bu44er6 (120&)
an* 6tore* 4roBen (<2!>C) an* 1rotecte* 4rom l-g5tM t5e6e 6olut-on6 65oul* be 6table 4or at lea6t 6-x mont56.
3n*-cator 6alt are orange re* 6ol-* an* 6oluble -n 'M() an* $ater (12 0&)
"M esters 65oul* be recon6t-tute* -n an5.*rou6 *-met5.l6ul4ox-*e ('M()) t5en u6e* a6 6oon a6 1o66-ble
t5erea4ter ($-t5-n a $ee%) to a;o-* 5.*rol.6-6 $-t5 6ub6eCuent lo66 o4 cell loa*-ng ca1ac-t.. 'M() 6toc%
6olut-on6 o4 AM e6ter6 65oul* be 4roBen an* *e6-ccate* an* 1rotecte* 4rom l-g5t.
Po66-ble AM e6ter *egra*at-on ma. be a66a.e* a6 4ollo$6=
- 1re1are a 1GM 6olut-on -n calc-um-4ree bu44er.
- mea6ure t5e 4luore6cence -nten6-t. (exc-tat-on at ,8 nm8 em-66-on at 2! nm).
- a** GM Ca
29
4or Fluo-3 an* Fluo-,
- c5ec% 4luore6cence aga-n.
Not5 -nten6-t. rea*-ng6 65oul* be ;er. lo$. (-gn-4-cantl. -ncrea6e* 4luore6cence u1on calc-um a**-t-on (-.e.8
-n t5e 6econ* rea*-ng) -n*-cate6 1art-al 5.*rol.6-6 o4 t5e AM e6ter.
Fluo-
*uidelines (or use + cell loading
T5e 4ollo$-ng 1rotocol $a6 *e6-gne* 4or Fluo-3. 3t ma. be o1t-m-Be* *e1en*-ng on t.1e o4 Ca29 -n*-cator8
t.1e an* number o4 loa*e* cell68 t-me8 tem1erature are 6ome ot5er ex1er-mental 4actor6.
T5e $ater-6oluble 6alt 4orm6 o4 Ca
29
-n*-cator6 ma. be loa*e* -nto cell6 ;-a m-cro-nLect-on8 a**-t-on to 1atc5
1-1ette 6olut-on68 6cra1e loa*-ng8 or u6-ng 1-noc.tot-c cell-loa*-ng reagent.
T5e AM analog6 -n*-cator6 coul* be loa*e* *-rectl.=
1. Pre1are a 1-mM 6toc% 6olut-on -n 'M().
Oote= 3t -6 o4ten more con;en-ent an* e44ect-;e to a** t5e non--on-c *etergent Pluron-c
K
F-127 to get 4urt5er a better *-66olut-on o4 AM
-n*-cator= m-x t5e AM e6ter 6toc% 6olut-on -n 'M() $-t5 an eCual ;olume o4 2!P ($#;) Pluron-c
K
F-127 -n 'M() be4ore *-lut-on -n
t5e loa*-ng me*-um8 ma%-ng t5e 4-nal Pluron-c
K
F-127 concentrat-on about !.!2P.
2. Pre1are a 1@ GM $or%-ng 6olut-on -n t5e a11ro1r-ate bu44ere* 15.6-olog-cal me*-um.
Oote= A;o-* am-ne-conta-n-ng bu44er6 6uc5 a6 Tr-6.
Oote= T5e organ-c an-on-tran61ort -n5-b-tor6 1robenec-* (1@2. mM) or 6ul4-n1.raBone (!.1@!.2 mM) ma. be a**e* to t5e cell me*-um
to 4urt5er re*uce lea%age o4 t5e *e-e6ter-4-e* -n*-cator. (toc% 6olut-on6 o4 6ul4-n1.raBone an* 1robenec-* be-ng Cu-te al%al-neM rea*Lu6t
t5e 12 o4 me*-a a4ter a**-t-on.
3. 3ncubate* cell6 $-t5 t5e AM e6ter b. m-x-ng eCual ;olume6 4or 1@&! m-nute6 at 92!#937>C.
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FT-78932
Oote6= )ne can loa* a*5erent cell6 $-t5out l-4t-ng
7xact loa*-ng concentrat-on8 t-me an* tem1erature $-ll nee* to be *eterm-ne* em1-r-call.M -n general -t -6 *e6-rable to u6e t5e m-n-mum
*.e concentrat-on reCu-re* to .-el* 4luore6cence 6-gnal6 $-t5 a*eCuate 6-gnal to no-6e. (ubcellular com1artmental-Bat-on8 an -n5erent
1roblem $-t5 t5e AM e6ter loa*-ng tec5n-Cue8 -6 u6uall. le66ene* b. lo$er-ng t5e -ncubat-on tem1erature.
,. 'e-e6ter-4-cat-on = remo;e t5e AM e6ter 6olut-on b. $a65-ng -n -n*-cator-4ree me*-um an* -ncubate
cell6 3! m-nute6 to allo$ com1lete *e-e6ter-4-cat-on o4 -ntracellular AM e6ter6.
Oote= e;entual -n*-cator lea%age ma. be Cuenc5e* -4 nee*e* b. a**-t-on o4 ant--4luore6ce-n ant-bo*.
. Tetracarbox.late 4orm o4 t5e -n*-cator can be mea6ure* $-t5 %no$n Ca
29
concentrat-on6.
Calcul o( calcium concentration%
Calc-um concentrat-on an* 4luore6cence are relate* accor*-ng to t5e eCuat-on =
QCa
29
R:<
*
Q(F-F
m-n
)#(F
max
-F)R
F = 4luore6cence o4 t5e -n*-cator at ex1er-mental calc-um concentrat-on
F
m-n
= 4luore6cence -n t5e ab6ence o4 calc-um
F
max
= 4luore6cence o4 t5e -n*-cator at 6aturate* calc-um concentrat-on.
<
*
= ;ar. accor*-ng to a number o4 4actor6 -n cell6 -nclu*-ng 128 1rote-n6 concentrat-on68 -on-c 6trengt58
tem1erature an* ;-6co6-t.. Calibration o( the Kd 65oul* be nece66ar. 4or accurate mea6urement o4
-ntracellular calc-um concentrat-on6. (ee our %-t S Calc-um Cal-brat-on %-t T8 FP-2127A.
,ther technical in(ormation%
U Calc-um-b-n*-ng an* 61ectro6co1-c 1ro1ert-e6 o4 4luore6cent -n*-cator6 can ;ar. Cu-te mar%e*l. $-t5 6e;eral
1arameter68 a6 cellular en;-ronment.
- For exam1le8 Fluo-3 4luore6cence -n t5e nucleo1la6m 5a6 been 4oun* to be t$-ce t5at -n t5e c.to1la6m. 3n
6-tu re61on6e cal-brat-on6 o4 -ntracellular -n*-cator6 t.1-call. .-el* <* ;alue6 6-gn-4-cantl. 5-g5er t5an in vitro
*eterm-nat-on6.
- 2ea;. metal cat-on6 (e.g.8 Mn
29
8 Vn
29
8 Pb
29
) al6o -nter4ere $-t5 6e;eral -n*-cator68 6omet-me6 $-t5 5-g5er
a44-n-t. t5an Ca
29
. Perturbat-on6 to calc-um mea6urement6 cau6e* b. 1re6ence o4 t5e6e -on6 can be controlle* u6-ng
t5e 5ea;. metal-6elect-;e c5elator TP7O.
- 3n 6-tu cal-brat-on6 ma. t5u6 be reCu-re*. T5e. are 1er4orme* b. ex1o6-ng loa*e* cell6 to controlle* Ca
29
bu44er6 -n t5e 1re6ence o4 -ono15ore6 6uc5 a6 A-231878 ,-bromo A-23187 or -onom.c-n. An alternat-;e met5o* -6 to
6aturate t5e -ntracellular -n*-cator $-t5 Mn
29
b. a**-ng 2 mM Mn
29
to t5e extracellular me*-um -n t5e 1re6ence o4
-ono15ore.
- Far-at-on6 -n t5e -ntracellular -n*-cator concentrat-on bet$een cell6 or -n *-44erent cell com1artment6 a44ect
Cuant-tat-;e Ca
29
mea6urement6. Co-loa*-ng cell6 $-t5 a 1a-r o4 61ectrall. contra6te* -n*-cator6 (t.1-call. Fluo-3) can
g-;e an a11rec-at-on o4 6uc5 ;ar-at-on68 1ro;-*e* -n*-cator6 are eCuall. con6-6tent.
- Far-at-on6 o4 Ca
29
$-t5 -n t-me m-g5t al6o be Cue6t-one*8 but t5-6 -6 o4ten -n 4act t5e goal o4 t5e a66a.W
*uidelines (or use + screening assa-s .H/S0
All abo;e gu-*el-ne6 are 6u-table 4or 8,- an* 38,-$ell6 m-cro1late a66a.6 u6e* at great 6cale b.
15armaceut-cal 6creen-ng68 $-t5 4luor-meter 6uc5 a6 Molecular 'e;-ce6 FX3P+Y (Fluorometr-c 3mag-ng
Plate +ea*er).
Protocol ma. be mo*-4-e* 4or max-mum re6ult6 accor*-ng t5e cell t.1e.
Related products
TP7O= FP-,,73&A
Calc-um Cal-brat-on Nu44er <-t= 2127
Pluron-c
K
ac-* F-127= FP-373&1A
3onom.c-n= FP-3989A
A23187 4ree ac-*8 FP-283&2A
,-Nromo A23187 4ree ac-*8 FP-37222A
Fluo-8 AM8 , t-me6 br-g5ter t5an Fluo-3
CP7!2
Fluo-8 O/8 CZ2!
+5o*-, AM8 , t-me6 br-g5ter t5an +5o*-2
CD&!&2
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FluoProbes

FT-78932
References
- Chung K1.. [3n;ol;ement o4 Calc-um#Calmo*ul-n (-gnal-ng -n Cerco61or-n Tox-n N-o6.nt5e6-6 b. Cerco61ora n-cot-anae." Appl
Environ Microbiol 238 1187 (2!!3) Art-cle
- *u 4. et al8 [A1o1to6-6 o4 rat o6teobla6t6 -n 1roce66 o4 calc-4-cat-on -n ;-tro[ Acta Pharmacol Sin 28 8!8
-1
2 (2!!2)
- Heinemann ". et al8. [A no;el a66a. to mea6ure t5e calc-um 4lux -n 5uman ba6o15-l6= e44ect6 o4 c5emo%-ne6 an* ner;e gro$t5 4actor.[
Pharmacology 258 ,9 (2!!3)
- 6a((e! 7. et al8 8 T5e Pat5 o4 Calc-um -n C.to6ol-c Calc-um )6c-llat-on6= A An-4.-ng 2.1ot5e6-6T8 Proc. Natl. Acad. Sci. USA 998
9883 (1991) Art-cle
- 6os'in M. et al, [T5e X-t.1e ;oltage-*e1en*ent Ca29 c5annel 7?X
-1
9 control6 bo*. $all mu6cle 4unct-on -n Caenor5ab*-t-6 elegan6.[
J Cell Biol :#38 337 (2!!2) Art-cle
- Kao 6.P.;. et al.8 HP5otoc5em-call. generate* c.to6ol-c calc-um 1ul6e6 an* t5e-r *etect-on b. 4luo-3 The Jo!rnal o" Cell Biology8
22<8 8179 (1989) Art-cle
- Kao! 6.P.;. et al, HAct-;e -n;ol;ement o4 Ca29 -n m-tot-c 1rogre66-on o4 ($-66 3T3 4-brobla6t6 T8 he Jo!rnal o" Cell Biology. 1118
183-19& (199!) Art-cle
- Merritt! 6.=. et al. Biochem. J. 2238 13(199!) Ab6tract
- Muschol M. et al, #Ca44e-ne -nteract-on $-t5 4luore6cent calc-um -n*-cator *.e6T Biophy$ Z 55! 77 (1999) Art-cle
- $a>atani K. et al, # Calc-um *-44u6-on coe44-c-ent -n ro* 15otorece1tor outer 6egment6 T Biophy$ J 928 728 (2!!2) Art-cle
- $iggli! =. et al8 S +eal-t-me con4ocal m-cro6co1. an* calc-um mea6urement6 -n 5eart mu6cle cell6= to$ar* t5e *e;elo1ment o4 a
4luore6cence m-cro6co1e $-t5 5-g5 tem1oral an* 61at-al re6olut-on. T Cell Calci!m 118 121(199!) Ab6tract
- Schroeder et al, J. Biomol. Screening 1(2)8 7 (199&).
- Schuman! M.". et al. S +ecomb-nant 5uman tumor necro6-6 4actor al15a -n*uce6 calc-um o6c-llat-on an* calc-um-act-;ate* c5lor-*e
current -n 5uman neutro15-l6. T5e role o4 calc-um#calmo*ul-n-*e1en*ent 1rote-n %-na6e J. Biol. Chem. 2&88 213,(1993) Art-cle
- ?uc>er! 1.S. et al8 H744ect6 o4 15otolab-le calc-um c5elator6 on 4luore6cent calc-um -n*-cator6. T8 Cell Calci!m. :8 29 (1992)
Ordering information
Catalog 6-Be Cuant-te6 an* 1r-ce6 ma. be 4oun* at 5tt1=##$$$.-nterc5-m.com
Plea6e -nCu-re 4or 5-g5er Cuant-t-e6 (a;a-b-l-t.8 65-1ment con*-t-on6).
Fluo-38 AM e6ter FP-78932A 1 mg
Fluo-38 AM e6ter FP-78932N 1!x1!! Gg
Fluo-38 AM e6ter FP-78932C 2!x! Gg
Fluo-38 AM e6ter FluoroPure gra*e FP-R1245A 2!x! Gg
Fluo-38 AM e6ter FluoroPure gra*e FP-R1245B mg
Fluo-38 AM e6ter FP-M2!3&A 1 ml 1mM -n *r. 'M()
Fluo-38 1entaammon-um 6alt FP-37!2!A 1 mg
Fluo-38 < 6alt FP-!3&&9A 1 mg
Fluo-38 Oa 6alt FP-33!21A 1 mg
For an. -n4ormat-on8 1lea6e a6% = Fluo1robe6 # 3nterc5-mM 2otl-ne = 933(!), 7! !3 73 !&
@isclaimer % Mater-al6 4rom FluoProbe6
K
are 6ol* (or research use onl-8 an* are not -nten*e* 4or 4oo*8 *rug8 5ou6e5ol*8 or co6met-c u6e.
FluoProbe6
K
-6 not l-able 4or an. *amage re6ult-ng 4rom 5an*l-ng or contact $-t5 t5-6 1ro*uct.
Pluron-c
K
F-127 -6 a reg-6tere* tra*emar% o4 NA(F.
<oLo
TM
an* XoLo
TM
are t5e reg-6tere* tra*emar% o4 Te4lab6.
+e;.F!1FN
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Fluo1robe68 1o$ere* b. P.,