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Live or Dead™ Fixable Dead Cell Staining Kit *NIR Fluorescence*

Our Live or Dead™ Fixable Dead Cell Staining 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 fixed mammalian cells in infrared fluorescence for flow cytometry applications with red laser excitation. The kit uses a proprietary red fluorescent dye that is more fluorescent upon binding to cellular components. The fluorescent dye used in the kit is well excited with the red (635 nm) to fluorescence at 775 nm (APC/CY7 channel). The kit provides all the essential components with an optimized cell-labeling protocol. It is an excellent tool for preserving of fluorescent images of particular cells, and can also be used for fluorescence flow cytometry applications.

Example protocol

AT A GLANCE

Protocol summary

  1. Prepare samples in HHBS (0.5 mL/assay)
  2. Replace with HHBS
  3. Add Stain It™ NIR fluorescence to the cell suspension
  4. Stain the cells at room temperature or 37°C for 20 - 60 minutes
  5. Wash the cells
  6. Fix the cells (optional)
  7. Examine the sample with flow cytometer and/or fluorescence microscope using the appropriate Excitation/Emission filter 

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

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. Stain It™ NIR fluorescence stock solution (500X):
Add 200 µL DMSO (Component B) into the vial of Stain It™ NIR fluorescence (Component A) to make 500X Stain It™ NIR fluorescence stock solution.

SAMPLE EXPERIMENTAL PROTOCOL

Table 1. Fluorescence spectra properties and suggested excitation laser for flow cytometry analysis

Cat. #DescriptionEx (nm)Em (nm)Excitation Source
22500Blue Fluorescence with 405 nm Excitation410450405 nm
22501Green Fluorescence with 405 nm Excitation408512405 nm
22502Orange Fluorescence with 405 nm Excitation398550405 nm
22599Red Fluorescence Optimized for Flow Cytometry523617488 nm
22600Blue Fluorescence353442335 nm
22601Green Fluorescence498521488 nm
22602Orange Fluorescence547573561 nm or 488 nm
22603Red Fluorescence583603 561 nm
22604Deep Red Fluorescence649660633 nm
22605Near Infrared Fluorescence749775633 nm 
  1. Prepare cells using 1X Hanks and 20 mM Hepes buffer (HHBS) or sodium azide-free and serum/protein-free buffer of your choice.

  2. Wash cells once with HHBS or the azide- and serum/protein-free buffer of your choice.

  3. Resuspend cells at 5 - 10 × 106/mL in HHBS or in the azide- and serum/protein-free buffer of your choice.

  4. Add 1 µL of 500X Stain It™ NIR fluorescence stock solution to 0.5 mL of cells/assay and mix it well.

  5. Incubate at room temperature or 37°C, 5% CO2 incubator for 20 - 60 minutes, protected from light. Note: The optimal stain concentrations and incubation time should be experimentally determined for different cell lines.

  6. Wash cells twice and resuspend cells with HHBS or the buffer of your choice.

  7. Fix cells as desired (optional).

  8. Analyze cells with flow cytometer and/or fluorescence microscope using the appropriate Excitation/Emission filter (see Table 1).

Spectrum

Citations

View all 4 citations: Citation Explorer
$\alpha$$\beta$-T-cell receptor transduction gives superior mitochondrial function to $\gamma$$\delta$-T cells with promising persistence
Authors: Ishihara, Mikiya and Miwa, Hiroshi and Fujiwara, Hiroshi and Akahori, Yasushi and Kato, Takuma and Tanaka, Yoshimasa and Tawara, Isao and Shiku, Hiroshi
Journal: iScience (2023)
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 3, 2024

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Catalog Number22605
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Additional ordering information

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Spectral properties

Excitation (nm)

757

Emission (nm)

779

Storage, safety and handling

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

Platform

Flow cytometer

Excitation640 nm laser
Emission780, 60 nm filter
Instrument specification(s)APC-Cy7 channel

Fluorescence microscope

Excitation749 nm
Emission775 nm
Recommended plateBlack wall, clear bottom

Components

Detection of Jurkat cell viability by Live or Dead™ Fixable Dead Cell Staining Kits (Cat#22605). Jurkat cells were treated and stained with Stain It™ NIR, and then fixed in 3.7% formaldehyde and analyzed by flow cytometry. Live (Red), heat-treated (Green) and unstained (Blue) cells were distinguished with APC-Cy7 channel. 
Detection of Jurkat cell viability by Live or Dead™ Fixable Dead Cell Staining Kits (Cat#22605). Jurkat cells were treated and stained with Stain It™ NIR, and then fixed in 3.7% formaldehyde and analyzed by flow cytometry. Live (Red), heat-treated (Green) and unstained (Blue) cells were distinguished with APC-Cy7 channel. 
Detection of Jurkat cell viability by Live or Dead™ Fixable Dead Cell Staining Kits (Cat#22605). Jurkat cells were treated and stained with Stain It™ NIR, and then fixed in 3.7% formaldehyde and analyzed by flow cytometry. Live (Red), heat-treated (Green) and unstained (Blue) cells were distinguished with APC-Cy7 channel.