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Fluo-8® Green Calcium Indicators The Brightest Ca2+ Signal at 488 nm Excitation
In this issue
Fura-8™ Ratiometric Calcium Indicators
Non-Radioactive Glucose Uptake Assays
Multicolor Live or Dead™ Viability Assays for Flow Cytometry
Colorimetric Quantitation of Ammonia
Fluorimetric Quantitation of Formaldehyde
Fluorimetric Quantitation of Lactate Dehydrogenase
Fluorimetric Measurement of Glucose-6-Phosphate
Detection of Reactive Oxygen Species (ROS) Using Multicolor ROS Brite™ Reagents
Fluo-8® Green Calcium Indicators The Brightest Ca2+ Signal at 488 nm Excitation
Reporter Gene Analysis Gaussia Luciferase Reporter Gene Assay
Reporter Gene Analysis Firefly Luciferase Reporter Gene Assay
iFluor™ 594, a New Bright Fluorescent Labeling Dye an Excellent Replacement to Texas Red®, DyLight™ 594 and Alexa Fluor® 594 Dyes
trFluor™ Fluorescent Labeling Dyes and Their Bioconjugates *Optimized for TR-FRET Applications*
ReadiLink™ iFluor™ Antibody Labeling Kits
AssayWise Letters 2013, Vol. 2(1)
Carbachol dose responses were measured in HEK-293 cells with Fluo-8® AM 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 removed, and the cells were incubated with 100 µL of dye-loading solution containing Fluo-8® AM or Fluo-4 AM for 1 hour at room temperature. Carbachol (25 L/well) was added by NOVOstar to achieve the final indicated concentrations. The fluorescence signals were measured at Ex/Em = 490/525 nm. The EC50 of Fluo-8® AM is about 1.2 µM.
Key Features of Fluo-8® AM:
- Faster, more readily loaded into cells than Fluo-3 AM and Fluo-4 AM. Only room temperature is required.
- Brighter, much brighter than Fluo-3 AM and Fluo-4 AM in cells.
- Convenient, almost identical spectra to those of Fluo-4 AM.
U2OS cells were seeded overnight at 40,000 cells per 100 µL per well in a Costar black wall/clear bottom 96-well 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 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 1. Fluo-8® green fluorescent calcium indicators for live cell calcium imaging.
| Indicator ▲ ▼ | Ex (nm) ▲ ▼ | Em (nm) ▲ ▼ | Kd¹ ▲ ▼ | Φ² ▲ ▼ | FCa/FFree³ ▲ ▼ | Unit Size ▲ ▼ | Cat No. ▲ ▼ |
| Fluo-8®, AM | 495 | 516 | 389 nM | 0.16 | ∼200 fold | 1 mg | 21080 |
| Fluo-8®, AM | 495 | 516 | 389 nM | 0.16 | ∼200 fold | 10x50 µg | 21082 |
| Fluo-8®, AM | 495 | 516 | 389 nM | 0.16 | ∼200 fold | 20x50 µg | 21083 |
| Fluo-8®, AM | 495 | 516 | 389 nM | 0.16 | ∼200 fold | 5x50 µg | 21081 |
| Fluo-8H™, AM | 495 | 516 | 232 nM | 0.16 | ∼200 fold | 10x50 µg | 21091 |
| Fluo-8H™, AM | 495 | 516 | 232 nM | 0.16 | ∼200 fold | 1 mg | 21090 |
| Fluo-8L™, AM | 495 | 516 | 1.9 µM | 0.16 | ∼200 fold | 1 mg | 21096 |
| Fluo-8L™, AM | 495 | 516 | 1.9 µM | 0.16 | ∼200 fold | 10x50 µg | 21097 |
| Fluo-8FF™, AM | 495 | 516 | 10 µM | 0.16 | ∼200 fold | 10x50 µg | 21104 |
| Fluo-8FF™, AM | 495 | 516 | 10 µM | 0.16 | ∼200 fold | 1 mg | 21105 |
Recent Citations of Fluo-8® Calcium Indicators
- Andre M. Deslauriers, Amir Afkhami-Goli, Amber M. Paul, Rakesh K. Bhat, Shaona Acharjee, Kristofor K. Ellestad, Farshid Noorbakhsh, Marek Michalak, and Christopher Power. Neuroinflammation and Endoplasmic Reticulum Stress Are Coregulated by Crocin To Prevent Demyelination and Neurodegeneration. J. Immunol. 2011; 187: 4788 -4799.
- Christopher A. Del Negro, John A. Hayes, and Jens C. Rekling. Dendritic Calcium Activity Precedes Inspiratory Bursts in preBotzinger Complex Neurons. J. Neurosci. 2011; 31: 1017 - 1022.
- Ferdinand Maingat, Brendan Halloran, Shaona Acharjee, Guido van Marle, Deirdre Church, M. John Gill, Richard R. E. Uwiera, Eric A. Cohen, Jon Meddings, Karen Madsen, and Christopher Power. Inflammation and epithelial cell injury in AIDS enteropathy: involvement of endoplasmic reticulum stress. FASEB J. 2011; 25: 2211 - 2220.
- Jens C. Rekling, Kristian H. R. Jensen, and Henrik Jahnsen. Spontaneous cluster activity in the inferior olivary nucleus in brainstem slices from postnatal mice. J. Physiol. 2012; 590: 1547 - 1562.
- Karin Persson and Jens C. Rekling. Population calcium imaging of spontaneous respiratory and novel motor activity in the facial nucleus and ventral brainstem in newborn mice. J. Physiol. 2011; 589: 2543 - 2558.
- Mako Kurogi, Megumi Miyashita, Yuri Emoto, Yoshihiro Kubo, and Osamu Saitoh. Green Tea Polyphenol Epigallocatechin Gallate Activates TRPA1 in an Intestinal Enteroendocrine Cell Line, STC-1. Chem Senses 2012; 37: 167 - 177.
- Man Hagiyama, Tadahide Furuno, Yoichiroh Hosokawa, Takanori Iino, Takeshi Ito, Takao Inoue, Mamoru Nakanishi, Yoshinori Murakami, and Akihiko Ito. Enhanced Nerve"Mast Cell Interaction by a Neuronal Short Isoform of Cell Adhesion Molecule-1. J. Immunol. 2011; 186: 5983 - 5992.
- Marc Aurel Busche, Xiaowei Chen, Horst A. Henning, Julia Reichwald, Matthias Staufenbiel, Bert Sakmann, and Arthur Konnerth. Critical role of soluble amyloid-β for early hippocampal hyperactivity in a mouse model of Alzheimer's disease. PNAS 2012; 109: 8740 - 8745.
- Md. Shahidul Islam, Calcium Signaling, in Advances in Experimental Medicine and Biology, Vol. 740, Springer, New York, 2012, pp 44-83. Michael J. Corey, Coupled Bioluminescence Assays, Methods, Evaluations and Applications. Wiley, New York. 2009, PP160-190.
- Nicholas B. Last, Elizabeth Rhoades, and Andrew D. Miranker. Islet amyloid polypeptide demonstrates a persistent capacity to disrupt membrane integrity. PNAS 2011; 108: 9460 - 9465.
- Nicholas M. Mellen and Deepak Mishra. Functional Anatomical Evidence for Respiratory Rhythmogenic Function of Endogenous Bursters in Rat Medulla. J. Neurosci. 2010; 30: 8383 - 8392.
- Satoru Torii, Kentaro Kobayashi, Masayuki Takahashi, Kasumi Katahira, Kenji Goryo, Natsuki Matsushita, Ken-ichi Yasumoto, Yoshiaki Fujii-Kuriyama, and Kazuhiro Sogawa. Magnesium Deficiency Causes Loss of Response to Intermittent Hypoxia in Paraganglion Cells. J. Biol. Chem. 2009; 284: 19077 - 19089.
- Shaona Acharjee, Yu Zhu, Ferdinand Maingat, Carlos Pardo, Klaus Ballanyi, Morley D. Hollenberg, and Christopher Power. Proteinaseactivated receptor-1 mediates dorsal root ganglion neuronal degeneration in HIV/AIDS. Brain 2011; 134: 3209 - 3221.
- Soichi Watanabe, Andre P. Seale, E. Gordon Grau, and Toyoji Kaneko. Stretch-activated cation channel TRPV4 mediates hyposmotically induced prolactin release from prolactin cells of Mozambique tilapiaOreochromis mossambicus. Am J Physiol Regulatory Integrative Comp Physiol. 2012; 302: R1004 - R1011.
- Takako Saito, Kogiku Shiba, Kazuo Inaba, Lixy Yamada, and Hitoshi Sawada. Self-incompatibility response induced by calcium increase in sperm of the ascidian Ciona intestinalis. PNAS 2012; 109: 4158 - 4162.
- Wei-Wei Shen, Maud Frieden, and Nicolas Demaurex. Local Cytosolic Ca2+Elevations Are Required for Stromal Interaction Molecule 1 (STIM1) De-oligomerization and Termination of Store-operated Ca2+ Entry. J. Biol. Chem.2011; 286: 36448 - 36459.
- Wen Fu, Araya Ruangkittisakul, David MacTavish, Jenny Y. Shi, Klaus Ballanyi, and Jack H. Jhamandas. Amyloid β (Aβ) Peptide Directly Activates Amylin-3 Receptor Subtype by Triggering Multiple Intracellular Signaling Pathways. J. Biol. Chem. 2012; 287: 18820 - 18830.
- Yasuto Yamaguchi, Xiao-Yan Du, Lei Zhao, John Morser, and Lawrence L. K. Leung. Proteolytic Cleavage of Chemerin Protein Is Necessary for Activation to the Active Form, Chem157S, Which Functions as a Signaling Molecule in Glioblastoma. J. Biol. Chem. 2011; 286: 39510 - 39519.
- Yohei Okubo, Hiroshi Sekiya, Shigeyuki Namiki, Hirokazu Sakamoto, Sho Iinuma, Miwako Yamasaki, Masahiko Watanabe, Kenzo Hirose, and Masamitsu Iino. Imaging extrasynaptic glutamate dynamics in the brain. PNAS 2010; 107: 6526 - 6531.