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

OverviewpdfSDSpdfProtocol


Excitation (nm)
757
Emission (nm)
779
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.

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


Component A: Stain It™ NIR fluorecence1 vial
Component B: DMSO1 vial (200 µL)

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. # Description Ex (nm) Em (nm) Excitation Source
22500 Blue Fluorescence with 405 nm Excitation 410 450 405 nm
22501 Green Fluorescence with 405 nm Excitation 408 512 405 nm
22502 Orange Fluorescence with 405 nm Excitation 398 550 405 nm
22599 Red Fluorescence Optimized for Flow Cytometry 523 617 488 nm
22600 Blue Fluorescence 353 442 335 nm
22601 Green Fluorescence 498 521 488 nm
22602 Orange Fluorescence 547 573 561 nm or 488 nm
22603 Red Fluorescence 583 603  561 nm
22604 Deep Red Fluorescence 649 660 633 nm
22605 Near Infrared Fluorescence 749 775 633 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


Open in Advanced Spectrum Viewer
spectrum

Spectral properties

Excitation (nm)757
Emission (nm)779

Citations


View all 3 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
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

Application notes


Annexin V