While some assays utilize antibodies to study cell health, proliferation, cell cycle or apoptosis, other types of experiments can rely on non-antibody based methods of assessment, often called non-antibody chemical probes. These are reagents that localize to an organelle or indicate health based on chemical characteristics like hydrophobicity, charge, size and enzymatic activation.

Below is a table to aid in reagent choice for cell health and proliferation labeling applications. Cell type suitability indicates whether this reagent labels live cells exclusively or whether it can also label the dead cells in a live cell culture or cells that have been previously fixed with buffers like paraformaldehyde or methanol. Sample suitability indicates whether this reagent can be used to label already fixed tissue sections or single cells in suspension or cell culture. Application indicates their suitability for being analyzed by flow cytometry, microscopy platforms or both. Fixation simply indicates whether the reagent, once used to label a cell sample, will be retained with a paraformaldehyde fixation.

 

      Cell Type Suitability Sample Suitability Application Fixation
Chemical Probe Imaging Equivalent Channel Subcellular Localization Live Dead/ Fixed Tissue Cell Culture/ Single Cells Flow Cytometry Microscopy Retention with PFA Treatment
MitoSpy™ Green FM FITC Mitochondria      
MitoSpy™ Orange CMTMRos Alexa Fluor® 555, PE Mitochondria    
MitoSpy™ Red CMXRos Alexa Fluor® 594 Mitochondria    
MitoSpy™ NIR DiIC1(5) Alexa Fluor® 647, APC Mitochondria      
Calcein-AM FITC Cytoplasm      
Calcein Violet-AM BV421™ Cytoplasm      
CFDA-SE FITC Cytoplasm  
Tag-it Violet™ BV421™ Cytoplasm  
Helix NP™ Blue Coumarin, BV421™ Nucleus    
Helix NP™ Green FITC Nucleus    
Helix NP™ NIR Alexa Fluor® 647, APC Nucleus    
CytoPhase™ Violet Coumarin, BV421™ Nucleus  
DRAQ5™ Alexa Fluor® 647, APC Nucleus  
DRAQ7™ Alexa Fluor® 700, APC/Cy7 Nucleus    
7-AAD PE/Cy5, PE/Dazzle™ 594 Nucleus    
Propidium Iodide PE/Cy5, PE/Dazzle™ 594 Nucleus    
DAPI Coumarin, BV421™ Nucleus    
Zombie Dyes Several formats available. Cell Surface or Cytoplasmic Primary Amines    
Apotracker™ Green FITC Phosphatidylserine    
Annexin V Several formats available. Phosphatidylserine      
Flash Phalloidin™ Green 488 FITC Actin     N/A
Flash Phalloidin™ Red 594 Alexa Fluor® 594 Actin     N/A

 

Zombie dyes, Tag-it™ Violet, and CFSE-DA can be used on live cells for tracking applications.

Cellular division for any cell type is dependent on the inherent function, location and the response of cells to repair, apoptosis, or death. To divide, cells must duplicate a copy of their DNA, increase mitochondrial density, and assemble/synthesize microtubules during interphase. G2 is a checkpoint stage of interphase where the cell has two sets of dsDNA and must commit to mitosis. Mitosis is the actual division stage where two daughter cells are created. There can be symmetrical or asymmetrical division depending on the cell and tissue type. Mitosis can be further subdivided into prophase, metaphase, anaphase, and telophase as the nucleus undergoes division and chromatids are pulled away from one another. After mitosis, the cell will undergo the G0/G1 phase when the cells rest to become ready for the next round of replication.

cycle

During G0/G1, S, and G2 phases, some fluorogenic nucleic acid stains selectively bind dsDNA stoichiometrically to give us measurements of DNA mass based on fluorescence intensity. Cell-permeant nucleic acid stains can be used in instances where cells will be analyzed live. Propidium IodideDAPIDRAQ5™DRAQ7™CytoPhase™ VioletHelix NP™ NIR,  Helix NP™ Blue, and Helix NP™ Green can all be used to stain fixed cells for cell cycle analysis. However, only CytoPhase™ Violet and DRAQ5™ can be used to assess DNA content of live cells.

 

For additional reagents (including antibodies) on cell cycle analysis or DNA replication, check here.

Live and Dead Cell Cycle Analysis

CytoPhase™ Violet

 

Live Ramos cells treated with 5 µM CytoPhase™ Violet dye for 90 minutes at 37°C. 

DRAQ5™

 

Live C57BL/6 mouse bone marrow cells were stained with DRAQ5™.
 

Helix NP™ NIR

 

C57BL/6 mouse thymus cells were fixed using 70% chilled ethanol. The cells were incubated for one hour at -20°C, washed, then stained with Helix NP™ NIR at 5 µM.

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