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Cell Explorer™ Live Cell Labeling Kit *Red Fluorescence*

Image of HeLa cells stained with Cell Explorer™ Live Cell Labeling Kit *Red Fluorescence* (Cat#22609)in a Costar black wall/clear bottom 96-well plate. Cells were stained with Calcein Deep Red™ for 30 minutes and image was acquired with fluorescence microscope using Cy5 filter.
Image of HeLa cells stained with Cell Explorer™ Live Cell Labeling Kit *Red Fluorescence* (Cat#22609)in a Costar black wall/clear bottom 96-well plate. Cells were stained with Calcein Deep Red™ for 30 minutes and image was acquired with fluorescence microscope using Cy5 filter.
Image of HeLa cells stained with Cell Explorer™ Live Cell Labeling Kit *Red Fluorescence* (Cat#22609)in a Costar black wall/clear bottom 96-well plate. Cells were stained with Calcein Deep Red™ for 30 minutes and image was acquired with fluorescence microscope using Cy5 filter.
Flow Cytometry Analysis of Jurkat cells stained with Cell Explorer™ Live cells Labeling Kit (Cat#22609). Jurkat cells were washed once with HH buffer and stained with Calcein Deep Red™ for 30 minutes at 37C incubator. Cells were then washed with HH buffer and resuspended in HH buffer. The fluorescence intensities of Live cells (healthy, Red) and Dead cells (treated in 55°C water bath for 30 minutes, Green) were measured with NovoCyte 3000 flow cytometer using blue laser APC emission channel.
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Telephone1-800-990-8053
Fax1-800-609-2943
Emailsales@aatbio.com
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Spectral properties
Excitation (nm)643
Emission (nm)663
Storage, safety and handling
H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22
UNSPSC12352200

OverviewpdfSDSpdfProtocol


Excitation (nm)
643
Emission (nm)
663
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 in red fluorescence. The kit uses a proprietary non-fluorescent dye that becomes strongly fluorescence upon entering into live cells. The dye is a hydrophobic compound that easily permeates intact live cells. The hydrolysis of the non-fluorescent substrate by intracellular esterases generates a strongly red fluorescent hydrophilic product that is well-retained in the cell cytoplasm. Cells grown in black-walled plates can be stained and quantified in less than two hours. The assay is more robust than the tetrazolium salt or Alarmar Blue™-based assays. It can be readily adapted for high-throughput assays in a wide variety of fluorescence platforms such as microplate assays, immunocytochemistry and flow cytometry. It is useful in a variety of studies, including cell adhesion, chemotaxis, multidrug resistance, cell viability, apoptosis and cytotoxicity. The kit provides all the essential components with an optimized cell-labeling protocol.

Platform


Fluorescence microscope

ExcitationCy5 filter set
EmissionCy5 filter set
Recommended plateBlack wall/clear bottom

Components


Example protocol


AT A GLANCE

Protocol Summary
  1. Prepare cells in growth medium
  2. Remove the medium
  3. Add Calcein Deep Red™ working solution (100 µL/well for 96-well plates or 25 µL/well for 384-well plates)

  4. Incubate cells at 37°C for 30 minutes to 2 hours
  5. Wash the cells
  6. Examine the specimen under under fluorescence microscope with Cy5 filter (Ex/Em = 646/660 nm)
Important Note

Thaw all the components at room temperature before starting the experiment.

CELL PREPARATION

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

PREPARATION OF STOCK SOLUTIONS

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

Calcein Deep Red™ stock solution

Add 20 µL of DMSO into the vial of Calcein Deep Red™ (Component A) and mix well to make Calcein Deep Red™ stock solution. Note: 20 µL of Calcein Deep Red™ stock solution is enough for 1 plate. Note: Unused Calcein Deep Red™ stock solution can be aliquoted and stored at < -20 °C for 2 weeks if the tubes are sealed tightly. Avoid repeated freeze-thaw cycles and protect from light.

PREPARATION OF WORKING SOLUTION

Add 20 µL of Calcein Deep Red™ stock solution into 10 mL of HHBS (Component B) and mix well to make Calcein Deep Red™ working solution. Protect from light.

SAMPLE EXPERIMENTAL PROTOCOL

  1. Remove the growth medium from the cell plates. Note: It is important to remove the growth medium in order to minimize the background fluorescence and increase the signal to background ratio.
  2. Add 100 µL/well (96-well plate) or 25 µL/well (384-well plate) Calcein Deep Red™ working solution into the cell plate.

  3. Incubate the cells in a 37°C, 5% CO2 incubator for 30 minutes to 2 hours.
  4. Remove the Calcein Deep Red™ working solution from the cells.

  5. Wash the cells with HHBS (Component B) for 2 to 3 times, and replace with HHBS.
  6. Image the cells using a fluorescence microscope with Cy5 filter (Ex/Em = 646/660 nm).

Spectrum


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spectrum

Spectral properties

Excitation (nm)643
Emission (nm)663

Product Family


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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