Calcium Assays

Intracellular Ca++ levels have become important indicators for the activation state of ion channels and G-protein coupled receptors as well as for the phases of apoptosis and cell injury. Though the respective kinetics and the absolute amounts of the Calcium levels are different for each of these physiological processes there are common ways for monitoring them. Luminescent labels like Aequorin as well as fluorescent ones are versatile and widely used solutions for microplate assays. Fura 2 and Indo-1 provide ratiometric readout thereby reducing effects caused by leaking or bleached dyes or varying assay conditions.

Product name λ \λ max. (a) λ \λ max. (a) MW (g·mol-1) exc em exc em Kd Ca2+ (nM) Applications Free Ca2+(nm) High Ca2+ (nm) Fluo-3 AM(b) 1129 503 / weak 505 / 526 390 . Most classical calcium indicator . No Wash step needed Fluo-8 AM 1000 490 / weak 490 / 514 389 . 4 times brighter than Fluo-3 . Loading at room temperature Fluo-8H AM 1100 490 / weak 490 / 514 232 . High Ca2+ concentration indicator Fluo-8L AM 1100 490 / weak 490 / 514 1 860 . Low Ca2+ concentration indicator . Red calcium indicator Rhod-4 AM 530 / weak 530 / 555 525 . 4 times brighter and 10 times larger windows assay than Rhod-2 . Loading at room temperature

. Leakage resistant form of Fura-2 Calcium Assays

Fura-PE3 AM 1258 335 / 495 380 / 495 250 . Ratio of reads with 2 different λex. . Avoids interference due to dye distribution and photobleaching . Leakage resistant form of Indo-1

Indo-PE3 AM 1266 338 / 480 338 / 410 260 . Ratio of reads with 2 different λem. . Avoids interference due to dye distribution and photobleaching

(a) after hydrolysis (b) AM ester are membrane-permeant and thus increases greatly cell loading that can be performed by simple incubation of the cells or tissue preparation in a buffer containing the AM ester. Pluronic® F-127, a mild non-ionic detergent, can facilitate AM esters loading. The AM esters themselves do not bind to Ca2+. However, once they have entered the cells, they are rapidly hydrolyzed by intracellular esterases into the parent Ca2+ compounds, thus becoming fully fluorescent upon binding to Ca2+. Many other ion indicators are available in our Interchim’ range of dye. Please contact us for specific application needs.

votre source d’innovations Tél 33 (0)4 70 03 88 55 - Fax 33 (0)4 70 03 82 60 - www.interchim.com 23 24 Calcium Assays The nextgenerationcalciumindicatorforautomatedscreening(HTS)applications ■ Fluo-8 Ionomycin, Ca λ Fluo-3 Description Ionomycin, Ca Probenecid, watersoluble Probenecid, Cellculturetested,tosuppress effluxofdyes Related products: Description » » Standard Ca ■ Fluo-3 (490/514 The » » » » » Kd =390nm λ Fluo-8 Fluo-8 NoWash Calcium Fluo-8 NoWash Calcium complex withcalcium)makeFluo-8NWanidealindicatorformeasurement ofcellularcalcium. which dye groups When Single λ exc. exc. /λ /λ Rapid dyeloading :at Increased signalintensity Performed in96or384-wellmicrotiter-plate Convenient androbust:Nowashstepneeded. Appropriate apparentCa Low compartmentalization Large dynamicrange Fluo-3 Fluo-8 that em. em. AM AM greatly =505/523nm :(Hydr.,Ca cells of stays nm), Fluo-8 NW stimulated 2+ 2+ 2+ increase ionophore ionophore concentrationindicator AM -NoWash AM inside high (No 2+ exc./em. NW ) :505/523nm;Kd(Ca Wash) sensitivity, cells are the Assay Kit,1%FBSMedium Assay Kit,Mediumremoval with fluorescence cleaved can and agonists, 2+

binding affinity cross and its Ca by fluorescence >100 cell the esterases, 2+ 2+ FP-78932A P/N : FP-78932D FP-M2036A FP-78932C FP-78932B FP-R1245A RT of ) =390nM;MW:1000 membrane. receptor Fluo-8 indicators (ratherthan37°CrequiredforFluo-4 times 1 2 is NW. fluorescence resulting signals greatly Fluo-4 Once The characteristics FP-53989B FP-53989A FP-288653 FP-288652 CP7501 CJ2551 CJ2550 CJ2561 CJ2560 P/N : enhanced in inside .com.com the a 1 mg Qty 50 mg 1 ml(1mMsolutioninDMSO) 20 x50µg 10 x100µg 1 mgFluoProbesPureGrade votre source d’innovations enhancement release negatively the cell, upon of of the intracellular charged binding its lipophilic (when long AM) wavelength fluorescent to 5 mg 1 mg 10 x150mg 10 x150mg 5 x50µg 100 plates 10 plates 100 plates 10 plates Qty it blocking calcium, calcium. forms a

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■ Fura-PE3 AM & Fura-2 AM Leakage resistant form of Fura-2, a popular Setrometric Ca2+ indicator

Fura-2 AM may be useful in microplate studies, where cell lines with different properties are compared or where screening treatments lead to differences in the number of cells or dye loading. Some of the limitations in the use of Fura-2 appear to be overcome by the use of glass bottom microplates (See page 78).

Reference : Robinson JA et al., Ratiometric and non-ratiometric Ca2+ indicators for the assessment of intracellular free Ca2+ in a breast cancer cell line using a fluorescence microplate reader, J Biochem Biophys Methods. 2004 Mar 31;58(3):227-37.

Fura-PE3 is an improved version of Fura-2, that reduces cell leakage and thus increases dye loading accuracy. Description P/N : Qty Fura-2 AM FP-42776A 1 mg FP-42776C 20 x 50 µg Representative experiment of angiotensin II FP-85312A 1 ml (1 mM) (ANG II)-evoked changes in the ratio of fura 2 fluoresence (340/380 nm) in adherent neonatal rat cardiomyocytes Fura-PE3 AM FP-AM603A 500 µg

(NRC). Primary cultures of NRC loaded with Fura 2 Calcium Assays were stimulated with increasing concentrations of ANG II (10 pM-1 µM) either in the absence (control ; A) or ■ Indo-1 AM presence of AA-861 (10 µM ; B) or MK-571 (100 nM ; C).

Indo-1 AM has a shift in the emission from 485 nm to 405 nm in the presence of calcium. Indo-1 AM can be used with a single argon-ion laser for excitation and to monitor two different emissions.

Description P/N : Qty Indo-1 AM FP-427755 500 mg Related product FP-42775A 20 x 50 µg Description P/N : Qty FP-98180A 1 ml (1 mM) Calcium Calibration kit FP-21527A 1 kit ® Related products : Pluronic F-127 FP-37361A 1 g ® 4-bromo A-23187, Ca2+ ionophore for UV light-excited dyes FP-372221 1 mg Pluronic F-127, 20% FP-69806A 1 ml A-23187 (calcimycin or Calcium Ionophore III) solution (in DMSO) FP-28362B 5 mg to equilibrate intracellular and extracellular [Ca2+]

Representative tracings from indo 1-loaded myocytes show simultaneous Ca2+ transients (top panels) and cell length (bottom panels). Myocytes were stimulated at 0.5 Hz after 6 h of no treatment (control) (A), LPS (10 ng/ml) (B), or LPS with ANG II (100 nM) (C).

■ Indo-PE3 AM Leakage resistant form of Indo-1

Description P/N : Qty Indo-PE3 AM FP-AM602A 500 µg

votre source d’innovations Tél 33 (0)4 70 03 88 55 - Fax 33 (0)4 70 03 82 60 - www.interchim.com 27 28 Viability & Cytotoxicity Assays or application At Several » » » » » Cell countingisrequired methods forgeneraltospecificcellassays. Interchim It maybeusefultoquantitateonlyviablecells,orfastproliferatingcells. Cell counting, &proliferation viability 51 Probe Selection guide Propidium Trypan blue Iodide, MTT Hoechst XTT UptiBlue WST Calcein-AM GAPDH CFSE Luciferin Syst. LDH AnnexinV BRDU -3H Thymidine

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