Calcium Detection Probes & Assay Kits

Calcium Detection Probes & Assay Kits 2016-2017

Cal-520™ Cal-590™ Fluo-8®

AAT Bioquest® Advancing Assay & Test Technologies Our Mission

AAT Bioquest® is committed to constantly meet or exceed its customer’s requirements by providing consistently high quality products and services, and by encouraging continuous improvements in its long-term and daily operations. Our core value is Innovation and Customer Satisfaction.

Our Story

AAT Bioquest®, Inc. (formerly ABD Bioquest, Inc.) develops, manufactures and markets bioanalytical research reagents and kits to life sciences research, diagnostic R&D and drug discovery. We specialize in photometric detections including absorption (color), fluorescence and luminescence technologies. The Company's superior products enable life science researchers to better under- stand biochemistry, immunology, cell biology and molecular biology. AAT Bioquest offers a rapidly expanding list of enabling products. Besides the standard catalog products, we also offer custom services to meet the distinct needs of each customer. Our current services include custom synthesis of biological detection probes, custom development of biochemical, cell-based and diagnostic assays and custom high throughput screening of drug discovery targets.

It is my greatest pleasure to welcome you to AAT Bioquest. We greatly appreciate the constant support of our valuable customers. While we continue to rapidly expand, our core value remains the same: Innovation and Customer Satisfaction. We are committed to being the leading provider of novel biological detection solutions. We promise to extend these values to you during the course of our service and to continue to support you with our new products and services. It is our greatest honor to receive valuable feedbacks and suggestions from you so that we can better serve your projects.

Very truly yours,

Zhenjun Diwu, Ph.D. President Table of Contents

General Information...... 2 Fluorescent Calcium Indicators...... 5 Fluo-8® Calcium Indicators...... 6 Cal-520™ Calcium Indicators...... 7 Cal-590™ Calcium Indicators...... 9 Cal-630™ Calcium Indicators...... 10 Rhod-4™ Calcium Indicators...... 11 Ratiometric Calcium Indicators...... 12 BTC...... 12 Fura-2...... 12 Fura-8™...... 12 Indo-1...... 13 FLIPR® Calcium Assays...... 14

1 General Information www.aatbio.com

Trademarks of AAT Bioquest Trademarks of Other Companies

AAT Bioquest® Alexa Fluor® (Life Technologies) Cal-520™ Calcium-Green™ (Life Technologies) Cal-520FF™ Cy3® (GE Healthcare) Cal-590™ FDSS® (Hamamatsu) Cal-630™ FlexStation® (Molecular Devices) Cal Green™ FLIPR® (Molecular Devices) Fluo-8® Texas Red® (Life Technologies)

Fluo-8FF™ Fluo-8H™ Fluo-8L™ Fura-8™ Rhod-4™ Screen Quest™ General Information

2 Tel: 800-990-8053 • Fax: 408-733-1304 Unless otherwise specified, all products are for Research Use Only. [email protected] • [email protected] Not for use in diagnostic or therapeutic procedures. www.aatbio.com General Information

CUSTOMER SERVICE & ORDERING INFORMATION

AAT Bioquest Corporate Headquarter: 520 Mercury Drive Sunnyvale, CA 94085, USA Phone: 800-990-8053 (US and Canada) 408-733-1055 (International) Fax: 408-733-1304 Website: www.aatbio.com E-mails: [email protected] (inquire) [email protected] (quote request) G [email protected] (technical support) ene r

International Distributors: al I See Back Cover n f o r m a tion

Tel: 800-990-8053 • Fax: 408-733-1304 3 Not for use in diagnostic or therapeutic procedures. [email protected] • [email protected] General Information www.aatbio.com

Custom Products and Services

Our Technologies Our Services Amplite™ enzyme-based detection platform is optimized for Besides the catalog products we also offer custom services to measuring horseradish peroxidase (HRP), alkaline phosphates, meet the distinct needs of each customer. Our current services luciferase, beta-galactosidase, lactamase, oxidase, protein include custom synthesis of biological detection probes, kinases, protein phosphatases, phosphodiesterases, proteases, custom development of biochemical, cell-based and diagnostic cytochrome P450, histone deacetylase (HDAC) and cell assays, custom bioconjugation and custom high throughput

signaling molecules such as NAD/NADH, NADP/NADPH, IP3, screening of drug discovery targets. cAMP and cGMP etc. Custom Assay Design and Development Cell Explorer™ cell labeling platform is a complete set of tools At AAT Bioquest we not only make probes and assay kits, but for tracking live cells. This platform is also widely used for also use them extensively ourselves. Scientists at AAT Bioquest sorting mixed populations of cells. are experts on assay design and have developed a wide variety Cell Navigator™ cell staining platform is a complete set of of tests that range from biochemical detection to cellular tools for selective labeling subcellular structures of live, fixed functions. Our assay options include: and dead cells. • Enzyme activities Cell Meter™ cellular functional assay platform is a complete set • Binding assays of tools for functional analysis of cellular events and real time- • Cell-based assays monitoring of cell functions. • Microplate assays iFluor™ superior fluorescent labeling dyes are optimized for • Flow cytometric analysis labeling proteins and nucleic acids. This group of dyes span • Fluorescence imaging from UV to infrared wavelength with good photostability and Custom Conjugation brightness.

General Information AAT Bioquest offers the best and the most rapid bioconjuga- mFluor™ superior fluorescent labeling dyes are optimized for tion service in the industry. flow cytometry applications. • Biotinylation PhosphoWorks™ detection platform is a set of tools for • Fluorescence labeling (iFluorTM, mFluor™, APC, RPE and detection of ATP, ADP, AMP, phosphate, pyrophosphate, PerCP) phosphoproteins and phosphopeptides. • Enzyme labeling (AP and HRP) Quest View™ colorimetric protease platform is a sensitive and • Small molecule conjugation robust tool for rapid detection of protease and glycosidase Custom Screening biomarkers. This technology platform has been licensed by a few diagnostic companies for developing rapid diagnostic AAT Bioquest offers on-demand high-throughput screening tests. and pharmacology profiling assays with multiple methodologies. Functional assays are designed, validated and RatioWorks™ superior cellular dyes are a sensitive and robust customized to the needs of our pharmaceutical and tool set for ratio imaging and real time monitoring of cellular biotechnology industry clients. These assays are aimed at functions (such as pH and ions) in live cells. assessing and monitoring the efficacy, tolerability and safety Screen Quest™ assay kits are a set of HTS-ready tools for high parameters of candidate compounds for treating and/or throughput screening of biochemical and cellular targets such diagnosing cancer, infectious disease, autoimmunity and as protein kinases, proteases, HDAC, cell apoptosis and transplantation. Our screening options include: cytotoxicity, GPCR, ion channels, ADME and transporters. • Full assay development for a target of your choice Tide Fluor™ and Tide Quencher™ superior labeling dyes are • Optimization of your assay protocol for HTS specially optimized for labeling nucleotides and peptides. • Multiple assay platforms and detection methods This platform offers the best value in the industry. It is second • Custom data analysis to none in terms of performance and cost. This technology platform has been licensed by a few diagnostic companies for Custom Synthesis of Fluorophores and Luminophores developing IVD diagnostic tests. AAT Bioquest is recognized by the top pharmaceutical trFluor™ superior fluorescent labeling dyes are optimized for companies and diagnostic companies as a key provider of developing time-resolved fluorescence-based assays. It has novel fluorescent dyes and luminescent probes. Over the years been used for developing HTS assay technologies for many we have developed and synthesized many enabling drug discovery targets. fluorescent and luminescent probes for running a variety of challenging biological detection tasks.

Tel: 800-990-8053 • Fax: 408-733-1304 Unless otherwise specified, all products are for Research Use Only. 4 [email protected] • [email protected] Not for use in diagnostic or therapeutic procedures. www.aatbio.com Fluorescent Calcium Indicators

Fluorescent Calcium Indicators

Calcium acts as a universal second messenger in a variety of cells. • Dissociation Constant (Kd): The desired indicators must have 2+ 2+ Numerous functions of all types of cells are regulated by Ca , thus a proper Kd compatible with the Ca concentration range of 2+ calcium measurement is critical for various biological investiga- interest. The Kd values of Ca indicators are dependent on many tions. Since the 1920s, scientists have attempted to measure Ca2+, factors, including pH, temperature, ionic strength, viscosity, protein 2+ 2+ but few were successful due to the limited availability of Ca binding, the presence of Mg and other ions. Consequently, Kd probes. The first reliable measurement of Ca2+ was performed by values for intracellular indicators are usually significantly higher Ridgway and Ashley by injecting the photoprotein aequorin into than the corresponding values measured in cell-free solutions. the giant muscle fiber of the barnacle. Subsequently, in the 1980s, Tsien and colleagues produced a variety of fluorescent indicators. Among the visible light-excitable calcium indicators, Fluo-8®, Among them Indo-1, Fura-2, Fluo-3 and Rhod-2 have been Fluo-4, Fluo-3, Rhod-2 and Rhod-4™ are most commonly used. 2+ the most valuable dyes for measuring Ca with a fluorescence Fluo-8® indicators are widely used in flow cytometry and confocal Fluorescent Calcium Indicators instrument. In recent years, AAT Bioquest has introduced the most laser-scanning microscopy. More recently, Fluo-8® AM has been robust calcium probes: Fluo-8® and Cal-520™, both of which enable extensively used for high throughput screening GPCR targets. the high throughput screening of GPCR and calcium channel drug Fluo-8® is essentially nonfluorescent unless bound to Ca2+ and discovery targets through the convenient calcium detection. FLIPR® exhibits a quantum yield of ~0.15 in the presence of saturating 2+ 2+ and FlexStation® instruments of Molecular Devices, FDSS®/μCELL Ca and a Kd of 390 nM for Ca . Cal-520™ is by far the best of Hamamatsu and NOVOstar of BMG Technologies have further 488 nm-excitable green fluorescent calcium indicator with a accelerated the high throughput measurement of calcium for GPCR significantly improved signal/background ratio and intracellular and ion channel research. retention.

Fluorescent probes that show spectral responses upon binding Ca2+ The long-wavelength Rhod-4™ is a valuable alternative Ca2+ have enabled researchers to investigate changes in intracellular indicator to the green fluorescent Fluo-8®, Fluo-4 and Fluo-3 for free Ca2+ concentrations by using fluorescence microscopy, flow experiments in cells and tissues that have high levels of autofluo- cytometry, fluorescence spectroscopy and fluorescence microplate rescence. Rhod-5N has a lower binding affinity for 2+Ca than any readers. Most of these fluorescent indicators are derivatives of other BAPTA-based indicator (Kd = ~320 µM) and is suitable for BAPTA chelators that incorporate a PET system responsive to Ca2+ measurements from 10 µM to 1 mM. Like the parent Rhod-2 calcium. There are quite a few factors that need be considered indicator, Rhod-5N is essentially nonfluorescent in the absence of when selecting a fluorescent Ca2+ indicator. These include: divalent cations and exhibits strong fluorescence enhancement with no spectral shift upon binding Ca2+. Both Fluo and Rhod • Spectral Properties: For UV excitation, Indo-1 and Fura-2 are indicators are available as cell-impermeant potassium salts or as widely used. Fura-8™ is a newly developed excitation-ratioable cell-permeant AM esters. calcium dye. Its AM is superior to Fura-2 AM with higher signal/ background ratio in cells. Fluo-8® and Cal-520™ are preferred for Table 1. Classic Single Wavelength Fluorescent Calcium Indicators 488 nm excitation while Cal-590™, Cal-630™, Rhod-2 and Rhod-4™ are used for red emissions. Ex Em Cat # Product Name Size K (nm) (nm) d • Measurement Mode: Ion indicators that exhibit spectral shifts Cal Green™-1 (equivalent to upon ion binding can be used for ratiometric measurements of 20500 10x50 µg 506 531 190 nM Calcium Green™-1) Ca2+ concentration, which are essentially independent of uneven Cal Green™-1 AM (equivalent to dye loading, cell thickness, photobleaching effects and dye 20501 10x50 µg 506 531 190 nM leakage. Excitation and emission wavelength preferences depend Calcium Green™-1 AM) on the type of instrumentation being used, as well as on sample 21011 Fluo-3 AM *UltraPure grade* 1 mg 506 526 390 nM autofluorescence and on the presence of other fluorescent or photoactivatable probes in the experiment. Indo-1, Fura-2 and 21018 Fluo-3, pentaammonium salt 1 mg 506 526 390 nM our newly developed Fura-8™ are primary choices for ratiometric 21017 Fluo-3, pentapotassium salt 1 mg 506 526 390 nM measurements while Fluo-3, Fluo-4, Fluo-8®, Cal-520™, Cal-590™, 21016 Fluo-3, pentasodium salt 1 mg 506 526 390 nM Cal-630™, Rhod-2 and Rhod-4™ are predominantly used for single wavelength measurements. 21064 Rhod-2 AM *UltraPure grade* 20x50 µg 549 578 570 nM 21067 Rhod-2, tripotassium salt 1 mg 549 578 570 nM • Permeability of Ca2+ Indicators (salt or AM ester): The salt forms are typically loaded into cells by microinjection, microprojectile 21068 Rhod-2, trisodium salt 1 mg 549 578 570 nM bombardment or electroporation, or used for extracellular assays. 21070 Rhod-5N AM 1 mg 551 577 0.3 mM In contrast, the cell-permeant acetoxymethyl (AM) esters can 21072 Rhod-5N, tripotassium salt 1 mg 551 577 0.3 mM be passively loaded into cells, where they are cleaved to cell- impermeant products by intracellular esterases.

Unless otherwise specified, all products are for Research Use Only. Tel: 800-990-8053 • Fax: 408-733-1304 5 Not for use in diagnostic or therapeutic procedures. [email protected] • [email protected] Fluo-8® Calcium Indicators www.aatbio.com

Fluo-8® Calcium Indicators

Fluo-3 and Fluo-4 were the most commonly used visible light- excitable calcium indicators. However, Fluo-3 AM and Fluo-4 AM are only moderately fluorescent in live cells upon esterase hydrolysis, and require harsh cell loading conditions to maximize their Fluo-8® AM cellular calcium responses. Fluo-8® dyes have been developed to improve cell loading and calcium response while maintaining the convenient Fluo-3 and Fluo-4 spectral wavelengths of maximum excitation @ ~490 nm and maximum emission @ ~520 nm. For cell loading, Fluo-8® AM only requires incubation at room temperature while Fluo-3 AM and Fluo-4 AM require incubation at 37 oC. In ad- Fluo-4 AM dition, Fluo-8® AM is 2 times brighter than Fluo-4 AM, and 4 times brighter than Fluo-3 AM in cells. AAT Bioquest offers a set of out- standing Fluo-8® reagents with different calcium binding affinities.

Carbachol Dose (μM) Key Features of Fluo-8® AM: Figure 1. Carbachol dose responses were measured in HEK-293 cells with Fluo-8® AM • Faster, more readily loaded into cells than Fluo-3 AM and (Cat# 21080) and Fluo-4 AM. HEK-293 cells were seeded overnight at 40,000 cells/100 µL/well in a 96-well black wall/clear bottom Costar plate. The growth medium was Fluo-4 AM. Only room temperature is required. removed, and the cells were incubated with 100 µL of dye-loading solution containing • Brighter, much brighter than Fluo-3 AM and Fluo-4 AM in cells. Fluo-8® AM or Fluo-4 AM for 1 hour at room temperature. Carbachol (25 µL/well) was added by NOVOstar to achieve the final indicated concentrations. The fluorescence

• Convenient, almost identical spectra to those of Fluo-4 AM. signals were measured at Ex/Em = 490/525 nm. The EC50 of carbachol measured with Fluo-8® AM is about 1.2 µM. Fluo-8® Calcium Indicators Calcium Fluo-8®

Fluo-3 AM Fluo-4 AM Fluo-8® AM

Figure 2. U2OS cells were seeded overnight at 40,000 cells per 100 µL per well in a Costar 96-well black wall/clear bottom plate. The growth medium was removed, and the cells were incubated with 100 µL of 4 µM Fluo-3 AM, Fluo-4 AM and Fluo-8® AM (Cat# 21080) in HHBS at 37 °C for 1 hour. The cells were washed twice with 200 µL HHBS, then imaged with a fluorescence microscope using FITC channel.

Table 2. Fluo-8® Calcium Indicators

Cat # Product Name Size Ex (nm) Em (nm) Kd 21080 Fluo-8® AM 1 mg 494 517 389 nM 21081 Fluo-8® AM 5x50 µg 494 517 389 nM 21088 Fluo-8®, sodium salt 10x50 µg 494 517 389 nM 21104 Fluo-8FF™ AM 10x50 µg 494 517 10 μM 21102 Fluo-8FF™, potassium salt 10x50 µg 494 517 10 μM 21090 Fluo-8H™ AM 1 mg 494 517 232 nM 21095 Fluo-8H™, sodium salt 10x50 µg 494 517 232 nM 21096 Fluo-8L™ AM 1 mg 494 517 1.9 μM 21098 Fluo-8L™, sodium salt 10x50 µg 494 517 1.9 μM

6 Tel: 800-990-8053 • Fax: 408-733-1304 Unless otherwise specified, all products are for Research Use Only. [email protected] • [email protected] Not for use in diagnostic or therapeutic procedures. www.aatbio.com Cal-520™ Calcium Indicators

Cal-520™ Calcium Indicators

Cal-520™ provides the most robust homogeneous fluorescence- based assay tool for detecting intracellular calcium mobilization. Cal-520™ AM is a new fluorogenic calcium-sensitive dye with a significantly improved signal to background ratio and intracellular retention compared to the existing green calcium indicators (such as Fluo-3 AM and Fluo-4 AM). The higher signal/background ratio and better intracellular retention make the Cal-520™ calcium assay a robust tool for evaluating GPCR and calcium channel targets as well as for screening their agonists and antagonists.

Our preliminary in-house research indicated that Cal-520™ AM can be readily loaded to a guinea pig heart and stays there for a few hours in the absence of probenecid. The calcium signal can be Cal-520™ Calcium Indicators readily monitored with Cal-520™ AM while it is difficult to observe ATP (μM) the calcium signal under the same conditions with other green Figure 3. ATP-stimulated calcium responses of endogenous P2Y receptor in CHO-K1 calcium dyes, such as Fluo-3 AM and Fluo-4 AM. cells incubated with Cal-520™ AM (red curve, Cat# 21131), or Fluo-4 AM (blue curve) respectively with probenecid under the same conditions. CHO-K1 cells were seeded overnight at 50,000 cells/100 µL/well in a Costar 96-well black wall/clear bottom plate. Table 3. Spectral Comparison of Fluo-3, Fluo-4, Fluo-8® and Cal-520™ 100 µL of 5 µM Fluo-4 AM or Cal-520™ AM in HHBS with 2.5 mm probenecid was added into the cells, and the cells were incubated at 37 °C for 2 hours. Dye Ex (nm) Em (nm) QY* Cal-520™ 492 514 0.75 Fluo-3 506 525 0.15 Fluo-4 493 515 0.16 Fluo-8® 490 514 0.16

*QY = Fluorescence Quantum Yield in the presence of 5 mM calcium citrate.

Key Features of Cal-520™ AM: • Better Intracellular Retention, Cal-520™ AM is better retained in live cells than Fluo-3 AM and Fluo-4 AM.

• Higher Sensitivity, Cal-520™ AM has much higher signal/ ATP (μM) background ratio than Fluo-3 AM and Fluo-4 AM in cells. Figure 4. ATP-stimulated calcium responses of endogenous P2Y receptors in CHO-K1 cells incubated with Cal-520™ AM (red curve, Cat# 21131), or Fluo-4 AM (blue curve) re- • Convenient, Cal-520™ AM has almost identical spectra to spectively, without probenecid under the same conditions. CHO-K1 cells were seeded those of Fluo-4 AM. overnight at 50,000 cells/100 µL/well in a Costar 96-well black wall/clear bottom plate. 100 µL of 5 µM Fluo-4 AM or Cal-520™ AM in HHBS was added into the cells, and the cells were incubated at 37 °C for 2 hours. Table 4. Fluorescent Cal-520™ Calcium Indicators

Cat # Product Name Size Ex (nm) Em (nm) Kd 21131 Cal-520™ AM 1 mg 492 514 320 nM 21141 Cal-520™, potassium salt 1 mg 492 514 320 nM 21136 Cal-520™, sodium salt 1 mg 492 514 320 nM 20606 Cal-520™-Biocytin Conjugate 5x50 μg 492 514 N/D 20605 Cal-520™-Biotin Conjugate 5x50 μg 492 514 N/D 20600 Cal-520™-Dextran Conjugate *MW 3,000* 1 mg 492 514 N/D 20601 Cal-520™-Dextran Conjugate *MW 10,000* 5 mg 492 514 N/D 20610 Cal-520™ Maleimide 100 μg 492 514 N/D 20609 Cal-520™ NHS Ester 100 μg 492 514 N/D 21142 Cal-520FF™ AM 1 mg 492 514 9.8 μM 21144 Cal-520FF™, potassium salt 10 x 50 µg 492 514 9.8 μM

Unless otherwise specified, all products are for Research Use Only. Tel: 800-990-8053 • Fax: 408-733-1304 7 Not for use in diagnostic or therapeutic procedures. [email protected] • [email protected] Cal-520™ Calcium Indicators www.aatbio.com

Recent Citations of Cal-520™

J.T. Lock et al., A comparison of fluorescent Ca2+ indicators for imaging local Ca2+ signals in cultured cells. Cell Calcium , 2015; doi: 10.1016/j.ceca.2015.10.003.

Kamran Melikov et al., Efficient entry of cell-penetrating peptide nona-arginine into adherent cells involves a transient increase in intracellular calcium. Biochemical Journal , 2015; 471: 221-230; doi: 10.1042/BJ20150272.

Wenxiang Hu et al., Direct conversion of normal and Alzheimer’s disease human fibroblasts into neuronal cells by small molecules. Cell Stem Cell, 2015; http://dx.doi.org/10.1016/j.stem.2015.07.006.

Carsten Tischbirek et al., Deep two-photon brain imaging with a red-shifted fluorometric Ca2+ indicator. PNAS, 2015; 112: 11377-11382; doi: 10.1073/pnas.1514209112. Liu, Y. et al., Solid-state sensors, actuators and microsystems (TRANSDUCERS), 2015 Transducers - 2015 18th Internation- al Conference on. 10.1109/TRANSDUCERS.2015.7180865.

Kyle L. et al., Spinning-spot shadowless TIRF microscopy. PLOS ONE, 2015; doi: 10.1371/journal.pone.0136055.

Songqing Tang et al., Extracellular calcium elicits feedforward regulation of the toll-like receptor-triggered innate immune response. Cellular & Molecular Immunology, 2015; doi: 10.1038/cmi.2015.59.

Angelo G. et al., Burst pacemaker activity of the sinoatrial node insodium–calcium exchanger knockout mice. PNAS , 2015; 112: 9769-9774. 10.1073/pnas.1505670112.

Søren Grubb et al., Preservation of cardiac function by prolonged action potentials in mice deficient of KChIP2. American Journal of Physiology - Heart and Circulatory Physiology, 2015; doi: 10.1152/ajpheart.00166.2015. Calcium Indicators ™ Calcium Cal-520 Sophie Payne et al., A critical role for the chromatin remodeller CHD7 in anterior mesoderm during cardiovascular development. Developmental Biology, 2015; 405: 82–95.

Emery Smith et al., Application of parallel multiparametric cell-based FLIPR detection assays for the identifica- tion of modulators of the muscarinic acetylcholine receptor 4 (M4). J Biomol Screen, 2015; 20: 858-868; doi: 10.1177/1087057115581770.

Christina Hanack et al., GABA blocks pathological but not acute TRPV1 pain signals. Cell, 2015; 160: 759–770.

He, Xi et al., Reduction of mitochondria–endoplasmic reticulum interactions by acetylcholine protects human umbilical vein endothelial cells from hypoxia/reoxygenation injury. Arteriosclerosis, Thrombosis, and Vascular Biology, 2015; AT- VBAHA-115.

Yuchen Hao et al., Development of automated patch clamp assay for evaluation of α7 nicotinic acetylcholine receptor agonists in automated QPatch-16. Assay and Drug Development Technologies, 2015; doi: 10.1089/adt.2014.622.

Vera Jansen et al., Controlling fertilization and cAMP signaling in sperm by optogenetics. eLife, 2015; 4: e05161.

Elena Revuelta-López et al., Hypoxia-driven sarcoplasmic/endoplasmic reticulum calcium ATPase 2 (SERCA2) downregula- tion depends on low-density lipoprotein receptor-related protein 1 (LRP1)-signalling in cardiomyocytes. Journal of Molecu- lar and Cellular Cardiology, 2015; 85: 25–36.

Lock JT et al., Imaging local Ca2+ signals in cultured mammalian cells. J Vis Exp., 2015; doi: 10.3791/52516.

Nasse, Jason S. et al., A novel slice preparation to study medullary oromotor and autonomic circuits in vitro. Journal of Neuroscience Methods, 2014; 237: 41-53.

Mass, Elvira et al., Murine creld1 controls cardiac development through activation of calcineurin/NFATc1 signaling. Devel- opmental Cell, 2014; 28: 711-726.

8 Tel: 800-990-8053 • Fax: 408-733-1304 Unless otherwise specified, all products are for Research Use Only. [email protected] • [email protected] Not for use in diagnostic or therapeutic procedures. www.aatbio.com Cal-590™ Calcium Indicators

Cal-590™ Calcium Indicators

Calcium measurement is critical for numerous biological investi- Cal-590™ has been developed to improve Rhod-2 AM cell loading gations. Fluorescent probes that show spectral responses upon and calcium response while maintaining the similar spectral wave- binding calcium have enabled researchers to investigate changes lengths of Rhod-2 AM, making it compatible with TRITC/Cy3® filter in intracellular free calcium concentrations by using fluorescence set. In CHO and HEK cells, the cellular calcium response of Cal-590™ microscopy, flow cytometry, fluorescence spectroscopy and fluo- is much more sensitive than that of Rhod-2 AM. The spectra of Cal- rescence microplate readers. Rhod-2 AM is most commonly used 590™ is well separated from those of FITC, Alexa Fluor® 488 and GFP, among the red fluorescent calcium indicators. However, Rhod-2 AM making it an ideal calcium probe for multiplexing intracellular as- is only moderately fluorescent in live cells upon esterase hydrolysis, says with GFP cell lines or FITC/Alexa Fluor® 488 labeled antibodies. and has very small cellular calcium responses. Cal-590™ Calcium Indicators

Figure 7. ATP-stimulated calcium response of endogenous P2Y receptor in CHO-K1 Figure 5. The excitation and emission spectra of Cal-590™ in the presence of calcium cells incubated with Cal-590™ AM (red curve, Cat# 20510) and Rhod-2, AM (blue curve) chloride (5 mM). under the same conditions. CHO-K1 cells were seeded overnight at the cell density of 50,000 cells per 100 μL per well in a 96-well black wall/clear bottom plate. 100 μL of 5 µg/mL Cal-590™ AM or Rhod-2 AM with 2.5 mM probenecid was added into the cells, and the cells were incubated at 37 oC for 1 hour. ATP (50 μL/well) was added by FlexStation® (Molecular Devices) to achieve the final indicated concentrations.

Control ATP

Figure 8. Responses of endogenous P2Y receptor to ATP in CHO-K1 cells. CHO-K1 cells were seeded overnight at 40,000 cells per 100 μL per well in a Costar 96-well black wall/clear bottom plate. 100 μL of 4 μM Cal-590™ AM (Cat# 20510) in HHBS with 1 mM probenecid were added into the wells, and the cells were incubated at 37 °C for 2 hours. The dye loading mediums were replaced with 100 μL HHBS and 1 mM Figure 6. Fluorescence emission spectra of Cal-590™ in solutions containing 0 to 39 probenecid , then imaged with a fluorescence microscope (Olympus IX71) using TRITC μM free Ca2+. channel before and after adding 50 μL of 300 μM ATP . Table 5. Cal-590™ Calcium Detection Indicators

Cat # Product Name Size Ex (nm) Em (nm) Kd (nM) 20510 Cal-590™, AM 5x50 μg 573 588 561 20511 Cal-590™, AM 10x50 μg 573 588 561 20512 Cal-590™, AM 1 mg 573 588 561 20518 Cal-590™, potassium salt 5x50 μg 573 588 561 20515 Cal-590™, sodium salt 5x50 μg 573 588 561 20508 Cal-590™-Dextran Conjugate *MW 3,000* 1 mg 573 588 N/D 20509 Cal-590™-Dextran Conjugate *MW 10,000* 1 mg 573 588 N/D

Unless otherwise specified, all products are for Research Use Only. Tel: 800-990-8053 • Fax: 408-733-1304 9 Not for use in diagnostic or therapeutic procedures. [email protected] • [email protected] Cal-630™ Calcium Indicators www.aatbio.com

Cal-630™ Calcium Indicators

X-Rhod-1 is commonly used as a red fluorescent calcium indicator. it compatible with Texas Red® filter set. In CHO and HEK cells, the However, X-Rhod-1 is only moderately fluorescent in live cells upon cellular calcium response of Cal-630™ is much more sensitive than esterase hydrolysis, and has very small cellular calcium responses. that of X-Rhod-1. The maximum emission wavelength of Cal-630™ In addition, X-Rhod-1 is mostly localized in mitochondria, thus is well separated from those of FITC, Alexa Fluor® 488 and GFP, mak- giving low signal/background ratio. Cal-630™ has been developed ing it an ideal calcium probe for multiplexing intracellular assays to improve X-Rhod-1 cell loading and calcium response while with GFP cell lines or FITC/Alexa Fluor® 488 labeled antibodies. maintaining the similar spectral wavelengths of X-Rhod-1, making

Figure 11. ATP-stimulated calcium response of endogenous P2Y receptor in CHO-K1 cells measured with Cal-630™ AM (Cat# 20530). CHO-K1 cells were seeded overnight at the cell density of 50,000 cells per 100 μL per well in a 96-well black wall/clear bottom Figure 9. Normalized emission spectra of Cal-520™ (Green), Cal-590™ (Orange) and plate. 100 μL of 10 µg/mL Cal-630™ AM with 2.0 mM probenecid was added into the Cal-630™ (Red). cells, and the cells were incubated at 37 ºC for 2 hours. ATP (50 μL/well) was added by FlexStation® (Molecular Devices) to achieve the final indicated concentrations. Calcium Indicators ™ Calcium Cal-630

Control ATP

Figure 12. Responses of endogenous P2Y receptor to ATP in CHO-K1 cells. CHO-K1 cells were seeded overnight at 40,000 cells per 100 μL per well in a 96-well black wall/clear bottom plate. 100 μL of 4 μM Cal-630™ AM (Cat# 20530) in HHBS with 1 mM probenecid were added into the wells, and the cells were incubated at 37 °C for 2 hours. The dye loading mediums were replaced with 100 μL HHBS and 1 mM probenecid , then imaged with a fluorescence microscope (Olympus IX71) using TRITC Figure 10. Fluorescence emission spectra of Cal-630™ in solutions containing 0 to 39 channel before and after adding 50 μL of 300 μM ATP . μM free Ca2+.

Table 6. Cal-630™ Calcium Detection Indicators

Cat # Product Name Size Ex (nm) Em (nm) Kd (nM) 20530 Cal-630™, AM 5x50 μg 608 626 792 20531 Cal-630™, AM 10x50 μg 608 626 792 20532 Cal-630™, AM 1 mg 608 626 792 20538 Cal-630™, potassium salt 5x50 μg 608 626 792 20535 Cal-630™, sodium salt 5x50 μg 608 626 792 20545 Cal-590™-Dextran Conjugate *MW 3,000* 1 mg 608 626 N/D 20546 Cal-590™-Dextran Conjugate *MW 3,000* 1 mg 608 626 N/D

10 Tel: 800-990-8053 • Fax: 408-733-1304 Unless otherwise specified, all products are for Research Use Only. [email protected] • [email protected] Not for use in diagnostic or therapeutic procedures. www.aatbio.com Rhod-4™ Calcium Indicators

Rhod-4™ Calcium Indicators

Rhod-2 is the most commonly used red fluorescent calcium detect calcium transients in stem cell cardiomyocytes that was not indicators. However, Rhod-2 AM is only moderately fluorescent detected with Rhod-2 AM under the same conditions. The higher in live cells upon esterase hydrolysis, and has very small cellular sensitivity of Rhod-4™ AM might be due to its higher cell loading calcium responses. Rhod-4™ has been developed to improve the efficiency than that of Rhod-2 AM. cell loading and calcium response while maintaining the spectral wavelength of Rhod-2. In CHO and HEK cells, the cellular calcium response of Rhod-4™ AM is 10 times more sensitive than that of Rhod-2 AM. Our in-house research indicated that Rhod-4™ AM can Rhod-4™ Calcium Indicators Rhod-4™

Carbachol Dose (μM)

Figure 13. The excitation and emission spectra of Rhod-4™ in PBS buffer (pH 7.2) in the presence of 5 mM calcium chloride. Figure 15. Carbachol dose responses were measured in HEK-293 cells with Rhod-4™ AM (red curve, Cat# 21120) and Rhod-2 AM (blue curve, Cat# 21064). HEK-293 cells were seeded overnight at 40,000 cells/100 μL/well in a Costar 96-well black wall/clear A B bottom 96-well plate. The growth medium was removed, and the cells were incubated with 100 μL Rhod-4™ AM dye loading solution, or 100 μL Rhod-2 AM dye loading Rhod-4™ AM solution (5 μM) for 1 hour at room temperature. Carbachol (25 μL/well) was added by NOVOstar (BMG Labtech) to achieve the final indicated concentrations. The EC50 of carbachol with Rhod-4™ AM was about 0.8 μM.

C D Recent Citations of Rhod-4™ Calcium Indicators Rhod-2 AM Caroline Arnold, Anja Feldner, Larissa Pfisterer, Maren Hödebeck, Kerstin Troidl, Guillem Genové, Thomas Wieland, Markus Hecker & Thomas Korff. RGS5 promotes arterial growth during arteriogen- esis. EMBO Molecular Medicine (2014) 6,1075-1089. DOI:10.15252/ Figure 14. ATP-stimulated calcium responses of endogenous P2Y receptors were emmm measured in CHO-K1 cells with Rhod-4™ AM (Cat# 21120) and Rhod-2 AM (Cat# 21064). CHO-K1 cells were seeded overnight at 50,000 cells/100 μL/well in a Costar 96-well black wall/clear bottom plate. The growth medium was removed, and the cells were Erwann Rousseau, Patrick P. Michel, and Etienne C. Hirsch. The Iron- incubated with 100 μL of dye loading solution using Rhod-4™ AM (4 µM, A and B) Binding Protein Lactoferrin Protects Vulnerable Dopamine Neurons or Rhod-2 AM (4 µM, C and D) for 1 hour in a 37 °C, 5% CO incubator. The cells were 2 from Degeneration by Preserving Mitochondrial Calcium Homeo- washed twice with 200 μL HHBS, then imaged before (A and C) and after (B and D) ATP treatment were taken with a fluorescence microscope (Olympus IX71) using TRITC stasis. Mol. Pharmacol., Dec 2013; 84: 888 - 898. channel.

Table 7. Visible Light-Excitable Orange Fluorescent Calcium Indicators

Cat # Product Name Size Ex (nm) Em (nm) Kd 21064 Rhod-2, AM *UltraPure grade" 20 x 50 µg 549 578 570 nM 21067 Rhod-2, tripotassium salt 1 mg 549 578 570 nM 21068 Rhod-2, trisodium salt 1 mg 549 578 570 nM 21120 Rhod-4™, AM 1 mg 524 551 451 nM 21128 Rhod-4™, sodium salt 5 x 50 µg 524 551 451 nM 21070 Rhod-5N, AM 1 mg 551 577 0.3 mM 21072 Rhod-5N, tripotassium salt 1 mg 551 577 0.3 mM

Unless otherwise specified, all products are for Research Use Only. Tel: 800-990-8053 • Fax: 408-733-1304 11 Not for use in diagnostic or therapeutic procedures. [email protected] • [email protected] Ratiometric Calcium Indicators www.aatbio.com

Ratiometric Calcium Indicators

BTC correlated to the amount of intracellular calcium. Regardless of the presence of calcium, Fura-2 emits at 510 nm. The use of the Among the ratiometric calcium indicators, Fura-2 and Indo-1 are ratio automatically cancels out confounding variables, such as most commonly used. BTC is another excitation-ratioable calcium variable dye concentration and cell numbers, making Fura-2 one indicator. However, BTC can only be used for high calcium level of the most appreciated tools to quantify calcium levels. Fura-2 is detection due to its low affinity to calcium. In recent years, BTC preferred for ratio-imaging microscopy, in which it is more practical has been increasingly used for monitoring potassium channels to change excitation wavelengths than emission wavelengths. since BTC demonstrated an excellent fluorescence enhancement response upon binding thallium ion that selectively passes through Fura-8™ potassium channels. Although Fura-2 has been widely used as the preferred excitation- ratioable calcium indicator, it has certain limitations, e.g., lower sensitivity compared to the single wavelength calcium dyes, such as Fluo-8® and Cal-520™. AAT Bioquest has recently developed Fura-8™ to improve the calcium response of Fura-2. As demonstrated in Figures 18 and 19, Fura-8™ AM is more sensitive to calcium than Fura-2 AM. In addition, Fura-8™ has its emission shifted to longer wavelength (Em = 525 nm). Fura-8™ might be also used for the flow cytometric analysis of calcium in cells due to its excellent excitation at 405 nm that perfectly matches the violet laser line.

Figure 16. The chemical structure of BTC AM (Cat# 21054). Key Features of Fura-8™: • Fura-8™ responses to calcium the same way as Fura-2 does

Ratiometric Indicators Calcium Fura-2 • Red-shifted dual excitation wavelengths (354 nm/415 nm) • Better excited at 405 nm for flow cytometric applications Fura-2 is a ratiometric fluorescent dye which binds free intracel- • Compatible with common filter sets lular calcium. It was the first widely-used dye for calcium imaging, and remains very popular. Fura-2 is excited at 340 nm and 380 • Higher signal/background ratio than that of Fura-2 nm, and the ratio of the emissions at those wavelengths is directly

Figure 18. Fluorescence excitation spectra of Fura-8™ in solutions containing 0 to Figure 17. Fluorescence excitation spectra of Fura-2 in solutions containing 0 to 39 µM free Ca2+. 39 µM free Ca2+.

12 Tel: 800-990-8053 • Fax: 408-733-1304 Unless otherwise specified, all products are for Research Use Only. [email protected] • [email protected] Not for use in diagnostic or therapeutic procedures. www.aatbio.com Ratiometric Calcium Indicators

lines of the argon-ion laser). The emission maximum of Indo-1 shifts from ~475 nm in Ca2+-free medium to ~400 nm when the 2+ Fura-8™ AM dye is saturated with Ca (see Figure 20). While Indo-1 is not cell permeant, its pentaacetoxymethyl ester, Indo-1 AM, enters the cell where it is cleaved by intracellular esterases to give Indo-1.

Fura-2 AM Ratiometric Calcium Indicators Ratiometric

Figure 19. ATP dose responses in CHO-K1 cells measured with Fura-2 AM (blue curve, Cat# 21021) and Fura-8™ AM (red curve, Cat# 21056) respectively. CHO-K1 cells were seeded overnight at 40,000 cells/100 μL/well in a Costar 96-well black wall/clear bottom plate. The cells were incubated with Fura-2 AM or Fura-8™ AM calcium assay dye-loading solution for 1 hour at room temperature. ATP (50 μL/well) was added by FlexStation®.

Indo-1

In contrast to Fura-2, Fura-8™ and BTC, Indo-1 is the preferred Figure 20. Fluorescence emission spectra of Indo-1 in solutions containing 0 to emission-ratioable dye for flow cytometry, where it is more practical 39 μM free Ca2+. to use a single laser for excitation (usually the 351–364 nm spectral

Table 8. Ratiometric Fluorescent Calcium Indicators

Zero Calcium High Calcium Cat # Product Name Size K Ex (nm) Em (nm) Ex (nm) Em (nm) d 21054 BTC, AM 1 mg 464 533 401 529 7 μM 21053 BTC, tetrapotassium salt 1 mg 464 533 401 529 7 μM 21021 Fura-2, AM *UltraPure grade* 1 mg 363 512 335 505 145 nM 21025 Fura-2, pentapotassium salt 1 mg 363 512 335 505 145 nM 21026 Fura-2, pentasodium salt 1 mg 363 512 335 505 145 nM 21055 Fura-8™, AM 1 mg 386 532 354 524 260 nM 21056 Fura-8™, AM 10x50 μg 386 532 354 524 260 nM 21057 Fura-8™, potassium salt 1 mg 386 532 354 524 260 nM 21058 Fura-8™, sodium salt 1 mg 386 532 354 524 260 nM 21032 Indo-1, AM *UltraPure grade* 1 mg 346 475 330 401 230 nM 21040 Indo-1, pentapotassium salt 1 mg 346 475 330 401 230 nM 21044 Indo-1, pentasodium salt 1 mg 346 475 330 401 230 nM 21050 Quin-2, AM 1 mg 353 495 333 495 60 nM 21052 Quin-2, tetrapotassium salt 5 mg 353 495 333 495 60 nM

Unless otherwise specified, all products are for Research Use Only. Tel: 800-990-8053 • Fax: 408-733-1304 13 Not for use in diagnostic or therapeutic procedures. [email protected] • [email protected] FLIPR® Calcium Assays www.aatbio.com

FLIPR® Calcium Assays

Calcium flux assays are preferred methods in drug discovery for Rhod-4™ AM is the brightest red calcium indicator available for HTS screening G protein coupled receptors (GPCRs). Cells expressing a screening. Once inside the cell, the lipophilic blocking groups of GPCR of interest that signals through calcium are pre-loaded with Rhod-4™ AM are cleaved by non-specific cell esterase, resulting in our proprietary Fluo-8® AM or Rhod-4™ AM which can cross cell a negatively charged fluorescent dye that stays inside cells, and its membrane. Screen Quest™ Calcium Assay Kits provide an opti- fluorescence is greatly enhanced upon binding to calcium. When mized assay method for monitoring GPCRs and calcium channels. cells stimulated with screening compounds, the receptor signals The assay can be performed in a convenient 96-well or 384-well the release of intracellular calcium, which greatly increases the microtiter-plate format and easily adapted to automation. fluorescence of Rhod-4™. The characteristics of its long wavelength, high sensitivity, and >250 times fluorescence increases (when it Fluo-8® AM is the brightest calcium indicator available for HTS forms complexes with calcium) make Rhod-4™ AM an ideal indica- screening. The characteristics of the convenient 488 nm excitation, tor for the measurement of intracellular calcium. high sensitivity, and 100-250 times fluorescence increases (when it forms complexes with calcium) make Fluo-8® AM an ideal indicator Screen Quest™ Fura-2 No Wash Calcium Assay Kit provides the for the measurement of intracellular calcium. only ratiometric FLIPR® calcium assay commercially available for screening GPCRs and calcium channel targets. The Fura-2 kit uses excitation ratio of 340/380 nm, monitoring emission at 510 nm.

Fluo-8® No Wash RFU (Max-Min) FLIPR® Calcium Assays FLIPR® Calcium Fluo-4 No Wash

Carbachol Dose (μM)

ATP (μM) Figure 21. Carbachol dose responses were measured in HEK-293 cells with Screen Quest™ Fluo-8® No Wash Calcium Assay Kit (blue curve, Cat# 36315) and Fluo-4 No Figure 22. ATP dose responses were measured in CHO cells with Screen Quest™ Fura-2 Wash Calcium Assay Kit (red curve). HEK-293 cells were seeded overnight at 40,000 No Wash Calcium Assay Kit (Cat# 36320). CHO-K1 cells were seeded overnight at 40,000 cells/100 μL/well in a Costar 96-well black wall/clear bottom 96-well plate. The cells cells/100 μL/well in a Costar 96-well black wall/clear bottom plate. The cells were were incubated with 100 μL of dye-loading solution using Screen Quest™ Fluo-8® incubated with 100 μL of Screen Quest™ Fura-2 No Wash Calcium Assay Kit for 1 hour No Wash Calcium Assay Kit or Fluo-4 No Wash Kit (according to the manufacturer’s at room temperature. ATP (50 μL/well) was added by FlexStation® (Molecular Devices) instructions) for 1 hour at room temperature. Carbachol (50 μL/well) was added by to achieve the final indicated concentrations. NOVOstar (BMG Labtech) to achieve the final indicated concentrations.

Table 9. Screen Quest™ FLIPR® Calcium Assay Kits

Cat # Product Name Size Ex (nm) Em (nm) 36315 Screen Quest™ Fluo-8® No Wash Calcium Assay Kit 10 plates 490 525 36316 Screen Quest™ Fluo-8® No Wash Calcium Assay Kit 100 plates 490 525 36308 Screen Quest™ Fluo-8® No Wash Calcium Assay Kit *Medium Removal* 10 plates 490 525 36309 Screen Quest™ Fluo-8® No Wash Calcium Assay Kit *Medium Removal* 100 plates 490 525 36320 Screen Quest™ Fura-2 No Wash Calcium Assay Kit 10 plates 340/380 510 36321 Screen Quest™ Fura-2 No Wash Calcium Assay Kit 100 plates 340/380 510 36334 Screen Quest™ Rhod-4™ No Wash Calcium Assay Kit 10 plates 530 590 36335 Screen Quest™ Rhod-4™ No Wash Calcium Assay Kit 100 plates 530 590 36331 Screen Quest™ Rhod-4™ No Wash Calcium Assay Kit *Medium Removal* 10 plates 530 590 36332 Screen Quest™ Rhod-4™ No Wash Calcium Assay Kit *Medium Removal* 100 plates 530 590

14 Tel: 800-990-8053 • Fax: 408-733-1304 Unless otherwise specified, all products are for Research Use Only. [email protected] • [email protected] Not for use in diagnostic or therapeutic procedures. www.aatbio.com Alphabetical Index

Alphabetical Index

PRODUCTBTC, AM NAME PAGE PRODUCT NAME PAGE BTC, AM 13 Fura-8™, AM 13 BTC, tetrapotassium salt 13 Fura-8™, potassium salt 13 Cal-520™, AM 7 Fura-8™, sodium salt 13 Cal-520™, potassium salt 7 Indo-1, AM *UltraPure grade* 13 Cal-520™, sodium salt 7 Indo-1, pentapotassium salt 13 Cal-520™-Biocytin Conjugate 7 Indo-1, pentasodium salt 13 Cal-520™-Biotin Conjugate 7 Quin-2, AM 13 Cal-520™-Dextran Conjugate *MW 3,000* 7 Quin-2, tetrapotassium salt 13 Cal-520™-Dextran Conjugate *MW 10,000* 7 Rhod-2, AM *UltraPure grade* 5, 11

Cal-520™ Maleimide 7 Rhod-2, tripotassium salt 5, 11 Alphabetical Index Cal-520™ NHS Ester 7 Rhod-2, trisodium salt 5, 11 Cal-520FF™ AM 7 Rhod-4™, AM 11 Cal-520FF™, potassium salt 7 Rhod-4™, sodium salt 11 Cal-590™, AM 9 Rhod-5N, AM 5, 11 Cal-590™, potassium salt 9 Rhod-5N, tripotassium salt 5, 11 Cal-590™, sodium salt 9 Screen Quest™ Fluo-8® No Wash Calcium Assay Kit 14 Cal-590™-Dextran Conjugate *MW 3,000* 9 Screen Quest™ Fluo-8® No Wash Calcium Assay Kit *Medium Removal* 14 Cal-590™-Dextran Conjugate *MW 10,000* 9 Screen Quest™ Fura-2 No Wash Calcium Assay Kit 14 Cal-630™, AM 10 Screen Quest™ Rhod-4™ No Wash Calcium Assay Kit 14 Cal-630™, potassium salt 10 Screen Quest™ Rhod-4™ No Wash Calcium Assay Kit *Medium Removal* 14 Cal-630™, sodium salt 10 Cal-630™-Dextran Conjugate *MW 3,000* 10 Cal-630™-Dextran Conjugate *MW 10,000* 10 Cal Green™-1 (equivalent to Calcium Green™-1) 5 Cal Green™-1 AM (equivalent to Calcium Green™-1 AM) 5 Fluo-3, AM *UltraPure grade* 5 Fluo-3, pentaammonium salt 5 Fluo-3, pentapotassium salt 5 Fluo-3, pentasodium salt 5 Fluo-8®, AM 6 Fluo-8®, sodium salt 6 Fluo-8FF™, AM 6 Fluo-8FF™, potassium salt 6 Fluo-8H™, AM 6 Fluo-8H™, sodium salt 6 Fluo-8L™, AM 6 Fluo-8L™, sodium salt 6 Fura-2, AM *UltraPure grade* 13 Fura-2, pentapotassium salt 13 Fura-2, pentasodium salt 13

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Catalog Number Index

CAT # PAGE CAT # PAGE

20500 5 21058 13 20501 5 21064 11 20508 9 21067 11 20509 9 21068 11 20510 9 21070 11 20511 9 21072 11 20512 9 21080 6 20515 9 21081 6 20518 9 21088 6 20530 10 21090 6 20531 10 21095 6 20532 10 21096 6 20535 10 21098 6 20538 10 21102 6 20545 10 21104 6 20546 10 21120 11 20600 7 21128 11 Catalog Number Index Catalog 20601 7 21131 7 20605 7 21136 7 20606 7 21141 7 20609 7 21142 7 20610 7 21144 7 21011 5 36308 14 21016 5 36309 14 21017 5 36315 14 21018 5 36316 14 21021 13 36320 14 21025 13 36321 14 21026 13 36331 14 21032 13 36332 14 21040 13 36334 14 21044 13 36335 14 21050 13 21052 13 21053 13 21054 13 21055 13 21056 13 21057 13

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