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Cell Explorer™ Live Cell Labeling Kit *Orange Fluorescence with 405 nm Excitation*

Fluorescence images of HeLa cells stained with Cell Explorer™ Live Cell Labeling Kits <em>Orange Fluorescence with 405 nm Excitation</em> (Cat#22616) in a Costar black wall/clear bottom 96-well plate. Cells were stained with CytoCalcein™ Violet 550 for 30 minutes. Image was aquired using fluorescence microscope.
Fluorescence images of HeLa cells stained with Cell Explorer™ Live Cell Labeling Kits <em>Orange Fluorescence with 405 nm Excitation</em> (Cat#22616) in a Costar black wall/clear bottom 96-well plate. Cells were stained with CytoCalcein™ Violet 550 for 30 minutes. Image was aquired using fluorescence microscope.
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Catalog Number22616
Unit Size
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Additional ordering information
Telephone1-408-733-1055
Fax1-408-733-1304
Emailsales@aatbio.com
InternationalSee distributors
ShippingStandard overnight for United States, inquire for international
Storage, safety and handling
H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22
UNSPSC12352200

OverviewpdfSDSpdfProtocol


Our Cell Explorer™ fluorescence imaging kits are a set of tools for labeling cells for fluorescence microscopic investigations of cellular functions. The effective labeling of cells provides a powerful method for studying cellular events in a spatial and temporal context. This particular kit is designed to uniformly label live cells for the flow cytometric analysis of live cells with the violet laser (405 nm excitation). The kit uses a proprietary dye that gets enhanced fluorescence upon entering into live cells. The dye is a hydrophobic compound that easily permeates intact live cells. The hydrolysis of the weakly fluorescent substrate by intracellular esterases generates a strongly fluorescent hydrophilic product that is well-retained in the cell cytoplasm. It can be readily adapted for flow cytometry applications. The fluorescent dye used in the kit is well excited with the violet laser (405 nm excitation) to fluorescence at ~550 nm. The kit provides all the essential components with an optimized cell-labeling protocol. It is useful for a variety of studies, including cell adhesion, chemotaxis, multidrug resistance, cell viability, apoptosis and cytotoxicity.

Platform


Flow cytometer

Excitation405 nm laser
Emission525/50 nm filter
Instrument specification(s)Pacific Orange channel

Fluorescence microscope

Excitation405 nm
Emission545/25 filter
Recommended plateBlack wall/clear bottom

Components


Component A: CytoCalcein™ Violet 5502 vials
Component B: HHBS 1 bottle (100 mL)

Example protocol


AT A GLANCE

Protocol summary

  1. Prepare cells in growth medium
  2. Remove growth medium
  3. Add CytoCalcein™ Violet 550 working solution (100 µL/well for a 96-well plate or 25 µL/well for a 384-well plate)
  4. Incubate the cells at 37°C for 30 minutes to 1 hour
  5. Wash the cells
  6. Examine the specimen under fluorescence microscope with 545/25 filter or flow cytometer with a 525/50 nm filter (Pacific Orange channel)

Important notes
Thaw all the components at room temperature before opening.

PREPARATION OF STOCK SOLUTION

Unless otherwise noted, all unused stock solutions should be divided into single-use aliquots and stored at -20 °C after preparation. Avoid repeated freeze-thaw cycles.

1. CytoCalcein™ Violet 550 stock solution:
Add 20 µL of DMSO into the vial of CytoCalcein™ Violet 550 (Component A) and mix well to make CytoCalcein™ Violet 550 stock solution. Note: 20 µL of CytoCalcein™ Violet 550 stock solution is enough for 1 plate. Note: Unused CytoCalcein™ Violet 550 stock solution can be aliquoted and stored at < -20 oC for one month if the tubes are sealed tightly. Avoid repeated freeze-thaw cycles and protect it from light.

PREPARATION OF WORKING SOLUTION

Add 20 µL of CytoCalcein™ Violet 550 stock solution into 10 mL of HHBS (Component B) and mix well to make CytoCalcein™ Violet 550 working solution.

For guidelines on cell sample preparation, please visit
https://www.aatbio.com/resources/guides/cell-sample-preparation.html

SAMPLE EXPERIMENTAL PROTOCOL

  1. Remove the growth medium.

  2. Add 100 µL/well (96-well plate) or 25 µL/well (384-well plate) of CytoCalcein™ Violet 550 working solution into the cell plate.

  3. Incubate the cells in a 37°C, 5% CO2 incubator for 30 minutes to 1 hour.

  4. Remove the CytoCalcein™ Violet 550 working solution from the cells, wash the cells with HHBS (Component B) for 2 to 3 times, and replace with HHBS.

  5. Analyze the cells using a fluorescence microscope with 545/25 filter or flow cytometer with a 525/50 nm filter (Pacific Orange channel).

Citations


View all 4 citations: Citation Explorer
Autophagy proteins are not universally required for phagosome maturation
Authors: Cemma, Marija and Grinstein, Sergio and Brumell, John H
Journal: Autophagy (2016): 1440--1446
Differential detection of tumor cells using a combination of cell rolling, multivalent binding, and multiple antibodies
Authors: Myung, Ja Hye and Gajjar, Khyati A and Chen, Jihua and Molokie, Robert E and Hong, Seungpyo
Journal: Analytical chemistry (2014): 6088--6094
Size control and biological properties of monodispersed mesoporous bioactive glass sub-micron spheres
Authors: Hu, Qing and Li, Yuli and Miao, Guohou and Zhao, Naru and Chen, Xiaofeng
Journal: Rsc Advances (2014): 22678--22687
Versatile fabrication of nanoscale sol--gel bioactive glass particles for efficient bone tissue regeneration
Authors: Lei, Bo and Chen, Xiaofeng and Han, Xue and Zhou, Jiaan
Journal: Journal of Materials Chemistry (2012): 16906--16913

References


View all 26 references: Citation Explorer
Requirements, features, and performance of high content screening platforms
Authors: Gough AH, Johnston PA.
Journal: Methods Mol Biol (2007): 41
A pharmaceutical company user's perspective on the potential of high content screening in drug discovery
Authors: Hoffman AF, Garippa RJ.
Journal: Methods Mol Biol (2007): 19
Optimizing the integration of immunoreagents and fluorescent probes for multiplexed high content screening assays
Authors: Giuliano KA., undefined
Journal: Methods Mol Biol (2007): 189
Past, present, and future of high content screening and the field of cellomics
Authors: Taylor DL., undefined
Journal: Methods Mol Biol (2007): 3
High-content fluorescence-based screening for epigenetic modulators
Authors: Martinez ED, Dull AB, Beutler JA, Hager GL.
Journal: Methods Enzymol (2006): 21
Application of laser-scanning fluorescence microplate cytometry in high content screening
Authors: Bowen WP, Wylie PG.
Journal: Assay Drug Dev Technol (2006): 209
High-content screening of known G protein-coupled receptors by arrestin translocation
Authors: Hudson CC, Oakley RH, Sjaastad MD, Loomis CR.
Journal: Methods Enzymol (2006): 63
Evaluation of a high-content screening fluorescence-based assay analyzing the pharmacological modulation of lipid homeostasis in human macrophages
Authors: Werner T, Liebisch G, Gr and l M, Schmitz G.
Journal: Cytometry A (2006): 200
Automated high content screening for phosphoinositide 3 kinase inhibition using an AKT 1 redistribution assay
Authors: Wolff M, Haasen D, Merk S, Kroner M, Maier U, Bordel S, Wiedenmann J, Nienhaus GU, Valler M, Heilker R.
Journal: Comb Chem High Throughput Screen (2006): 339
High concordance of drug-induced human hepatotoxicity with in vitro cytotoxicity measured in a novel cell-based model using high content screening
Authors: O'Brien P J, Irwin W, Diaz D, Howard-Cofield E, Krejsa CM, Slaughter MR, Gao B, Kaludercic N, Angeline A, Bernardi P, Brain P, Hougham C.
Journal: Arch Toxicol (2006): 580