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

Additional fluorescent color(s): 
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Detection of Jurkat cell viability by Live or Dead™ Fixable Dead Cell Staining Kits (Cat#22603). Jurkat cells were heat- treated at 60oC or left untreated, and stained with Stain It™ Red. Live and heat-treated cells were imaged with fluorescence microscope using Texas Red filter. 
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Unit Size: Cat No: Price (USD): Qty:
200 Tests 22603 $145


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Additional Ordering Information
Telephone: 1-800-990-8053
Fax: 1-408-733-1304
Email: sales@aatbio.com
International: See distributors





Overview

Ex/Em (nm)583/603
Storage Freeze (<-15 °C)
Minimize light exposure
InstrumentsFluorescence microscope, Flow cytometer
Category Cell Biology
Labeling Cells
Related Fluorescence Imaging
Our Live or Dead™ Fixable Dead Cell Staining Kits are a set of tools for labeling cells for fluorescence microscopic investigations of cell 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 red fluorescence for long term microscopic examination. The kit uses a proprietary red fluorescent dye that is more fluorescent upon bonding to cellular components. The fluorescent dye used in the kit is quite photostable so that the images can be repeatedly examined. 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 microscope demonstrations.




Protocol


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This protocol only provides a guideline, and should be modified according to your specific needs.
At a glance

Protocol summary

  1. Prepare samples in HHBS (0.5 mL/assay)
  2. Replace with HHBS
  3. Add Stain It™ Red 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.

Key parameters
Instrument:Fluorescence microscope
Excitation:583 nm
Emission:603 nm
Recommended plate:Black wall/clear bottom
  
Instrument:Flow cytometer
Instrument specification(s):See Table 1
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™ Red stock solution (500X):
Add 200 µL DMSO (Component B) into the vial of Stain It™ Red (Component A) to make 500X Stain It™ Red stock solution.

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

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 for flow cytometry staining 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™ Red 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).
Example data analysis and figures

Figure 1. Image of Hela cells fixed with formaldehyde and stained with Live or Dead™ Fixable Dead Cell Staining kit *Red Fluorescence* in a Costa black wall/clear bottom 96-well plate.
Disclaimer
AAT Bioquest provides high-quality reagents and materials for research use only. For proper handling of potentially hazardous chemicals, please consult the Safety Data Sheet (SDS) provided for the product. Chemical analysis and/or reverse engineering of any kit or its components is strictly prohibited without written permission from AAT Bioquest. Please call 408-733-1055 or email info@aatbio.com if you have any questions.





References & Citations

Autophagy proteins are not universally required for phagosome maturation
Authors: Marija Cemma, Sergio Grinstein, John H Brumell
Journal: Autophagy (2016): 1440--1446

Differential detection of tumor cells using a combination of cell rolling, multivalent binding, and multiple antibodies
Authors: Ja Hye Myung, Khyati A Gajjar, Jihua Chen, Robert E Molokie, Seungpyo Hong
Journal: Analytical chemistry (2014): 6088--6094

Versatile fabrication of nanoscale sol--gel bioactive glass particles for efficient bone tissue regeneration
Authors: Bo Lei, Xiaofeng Chen, Xue Han, Jiaan Zhou
Journal: Journal of Materials Chemistry (2012): 16906--16913

Advanced glycation end-products increase IL-6 and ICAM-1 expression via RAGE, MAPK and NF-$\kappa$B pathways in human gingival fibroblasts
Authors: K Nonaka, Y Kajiura, M Bando, E Sakamoto, Y Inagaki, JH Lew, K Naruishi, T Ikuta, K Yoshida, T Kobayashi
Journal: Journal of Periodontal Research






Additional Documents

 
Safety Data Sheet (SDS)


Catalogs
1. Cell Apoptosis & Proliferation

Application Notes
1. AssayWise Letters 2013, Vol 2(2)

Certificate of Analysis