Rapid identification of live and dead cells----- Exploring the mysteries of life

Rapid identification of live and dead cells----- Exploring the mysteries of life

The balance between cell proliferation and cell death in eukaryotic (mammalian cells) and prokaryotic (bacteria) cells is crucial for maintaining the organic development and life of cells. Imbalance can lead to various diseases. The state of live and dead cells is a common research focus in studying biological proliferation and growth. For example, in eukaryotic cells, the integrity of the cell membrane and intracellular esterase activity are analyzed to determine cell status using specific instruments, providing a convenient method for studying cell status and analyzing live and dead cells to assess cell vitality. It can be applied to most eukaryotic mammalian cells but is not suitable for fungi and yeast. It is mainly used for drug research, toxicity determination, and cell growth state research.

Arcegen Biology has meticulously developed a dual-stain detection reagent kit for eukaryotic and prokaryotic cells to meet your research needs.


Detection Principle

1. Cell staining

Calcein-AM is a commonly used fluorescent staining reagent for labeling live cells, emitting green fluorescence (Ex=490 nm, Em=515 nm). Derived from traditional Calcein (calcein green) by introducing the acetoxymethyl (AM) ester group, Calcein-AM increases hydrophobicity, allowing it to easily penetrate live cell membranes. Once inside the cell, Calcein-AM (which itself is non-fluorescent) is cleaved by intracellular esterases to form the membrane-impermeable polar molecule Calcein, which is retained within the cell and emits strong green fluorescence. Compared to other similar reagents (such as BCECF-AM and CFDA), Calcein-AM has extremely low cell toxicity, making it suitable as a fluorescent probe for live cell staining without inhibiting any cellular functions, such as proliferation and chemotaxis of lymphocytes.

Since dead cells lack esterases, Calcein-AM is only used for testing the cell viability of live cells and short-term labeling. Therefore, Calcein-AM is often used in combination with fluorescent probes for dead cells such as propidium iodide (PI) for simultaneous fluorescent double staining of live and dead cells. Propidium iodide (PI) cannot pass through the cell membrane of live cells and can only penetrate the disordered regions of the cell membrane of dead cells to reach the cell nucleus, where it embeds into the DNA double helix and produces red fluorescence (Ex=535 nm, Em=617 nm), thus PI only stains dead cells. Since both Calcein and PI-DNA can be excited at 490 nm, live cells and dead cells can be observed simultaneously under a fluorescence microscope. With excitation at 545 nm, only dead cells can be observed. This reagent kit is suitable for fluorescence microscopy, fluorescence microplate readers, flow cytometers, and other fluorescence detection systems.

2. Bacterial staining

The fluorescent dye DMAO is a green nucleic acid fluorescent dye that can stain both live and dead bacteria. EthD-III is a red nucleic acid fluorescent dye that can only stain dead bacteria with damaged cell membranes. When DMAO and EthD-III are mixed for staining, bacteria with intact cell membranes appear green, while bacteria with damaged cell membranes appear both green and red. The results can be analyzed using fluorescence microscopy or flow cytometry and are applicable to most types of bacteria.

The common standard for bacterial viability is the ability of bacteria to reproduce in appropriate nutrient media, known as growth determination. This reagent kit typically shows good consistency with growth determination results in liquid or solid media. Under certain conditions, bacteria with membrane damage may recover and reproduce in nutrient media, leading to a false positive in the viability assay. Conversely, some bacteria with intact membranes may not reproduce in nutrient media but may still be considered viable in the assay. Therefore, if there is a significant discrepancy between this assay and bacterial growth determination, the aforementioned possibilities should be considered.

Application Detection

Results of Use

1. Cell staining results

Note: Cell type = A375 melanoma cells, Calcein-AM = 2 μM, PI = 4.5 μM, staining time = 15 minutes

A is the Calcein-AM staining image, B is the PI staining image, C is the MERGE image

2. Bacterial staining results

 Note: Cell type = Escherichia coli, EthD-III = 2 ul, DMAO = 4 ul, Staining time = 15 minutes.

A is the EthD-III staining image, B is the DMAO staining image, and C is the MERGE image.


Related products

Product Name

Catalog Number


Calcein-AM/PI Double Stain Kit



Viability/Cytotoxicity Multiplex Assay Kit



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