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Cell Explorer™ Live Cell Tracking Kit *Orange Fluorescence*

Our Cell Explorer™ Live Cell Tracking Kit uses a proprietary orange fluorescent 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. The tracking dye has good photostability with robust imaging performance. The kit is particularly suitable for multicolor flow cytometric analysis of cells. It can also be used with a fluorescence microscope equipped with a TRITC filter set. This kit provides an effective tool of labeling cells for flow cytometric and fluorescence microscopic investigations of cellular functions. The effective labeling of cells offers a powerful method for studying cellular events in a spatial and temporal context. This kit is useful for 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.

Example protocol

AT A GLANCE

Protocol summary

  1. Prepare samples and remove cells from incubator
  2. Add 10 µL/well of 10X Track It™ Orange working solution in each well
  3. Stain the cells at 37°C for 15 minutes to 1 hour
  4. Wash the cells
  5. Examine the specimen under fluorescence microscope with Cy3/TRITC filter or flow cytometer with 575/26 nm filter (PE channel)

Important notes
Thaw all the components to room temperature, centrifuge the component A briefly before opening.

PREPARATION OF WORKING SOLUTION

Dilute 500X Track It™ Orange DMSO stock solution (Component A) into Assay Buffer (Component B) to make a 10X to 25X Track It™ Orange working solution. The working solution should be prepared enough for all the wells at 10 µL/well with the appropriate concentration. For example, to get a 1X final concentration of Track It™ Orange for one 96-well microplate, dilute 20 µL of the Track It™ Orange DMSO stock solution into 1 mL of Assay Buffer (Component B) to make 1 mL of 10X Track It™ Orange working solution. Note: The final concentration of the Track It™ Orange working solution should be empirically determined for different cell types and/or experimental conditions. It is recommended to test at the concentrations that are at least over a ten-fold range.

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

SAMPLE EXPERIMENTAL PROTOCOL

  1. Add 10X Track It™ Orange working solution to the cells, the volume should be equal to 1/10 of the volume of cell culture medium. For example, for a 96-well plate, add 10 µL/well of 10X Track It™ Orange working solution into the cells.

  2. Incubate the cells in a 37°C, 5% CO2 incubator for 15 minutes to 1 hour.

  3. Wash cells with Hanks and 20 mM Hepes buffer (HBSS) or an appropriate buffer.

  4. Fill the cell wells with growth medium.

  5. Analyze the cells using a fluorescence microscope with Cy3/TRITC filter sets of flow cytometer with 575/26 nm filter (PE channel).

Citations

View all 5 citations: Citation Explorer
Tricultured cell sheets develop into functional pancreatic islet tissue with a vascular network
Authors: Sekine, Hidekazu and Homma, Jun and Shimizu, Tatsuya
Journal: (2022)
Single Cell Bioprinting with Ultrashort Laser Pulses
Authors: Zhang, Jun and Byers, Patrick and Erben, Amelie and Frank, Christine and Schulte-Spechtel, Levin and Heymann, Michael and Docheva, Denitsa and Huber, Heinz P and Sudhop, Stefanie and Clausen-Schaumann, Hauke
Journal: Advanced Functional Materials (2021): 2100066
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
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
G protein-coupled receptor internalization assays in the high-content screening format
Authors: Haasen D, Schnapp A, Valler MJ, Heilker R.
Journal: Methods Enzymol (2006): 121
Page updated on November 9, 2024

Ordering information

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Unit size
Catalog Number22622
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Additional ordering information

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Storage, safety and handling

H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22
UNSPSC12352200

Platform

Flow cytometer

Excitation488 nm or 532 nm laser
Emission575, 26 nm filter
Instrument specification(s)PE channel

Fluorescence microscope

Excitation540 nm
Emission570 nm
Recommended plateBlack wall, clear bottom
Instrument specification(s)Cy3, TRITC filter

Components

Image of Hela cells stained with&nbsp;Cell Explorer&trade; Live Cell Labeling Kit *Orange Fluorescence<em>* </em>(Cat#22622)<em>&nbsp;</em>in 96-well Costar black wall/clear bottom. Cells were stained with&nbsp;Track It&trade; Orange for 15 minutes and image was aquired with fluorescence microscope using TRITC filter.
Image of Hela cells stained with&nbsp;Cell Explorer&trade; Live Cell Labeling Kit *Orange Fluorescence<em>* </em>(Cat#22622)<em>&nbsp;</em>in 96-well Costar black wall/clear bottom. Cells were stained with&nbsp;Track It&trade; Orange for 15 minutes and image was aquired with fluorescence microscope using TRITC filter.
Image of Hela cells stained with&nbsp;Cell Explorer&trade; Live Cell Labeling Kit *Orange Fluorescence<em>* </em>(Cat#22622)<em>&nbsp;</em>in 96-well Costar black wall/clear bottom. Cells were stained with&nbsp;Track It&trade; Orange for 15 minutes and image was aquired with fluorescence microscope using TRITC filter.