Fluorescent dye works differently from trypan blue staining dye. When fluorescent dye penetrates through the cell and binds to nucleic acid, it emits certain fluorescent signal at certain wavelength. To go into further detail, fluorescent dye can be distinguished into two types – membrane permeant and impermeant.
As fluorescent dye stains nucleic acid of cell, it provides more accurate viability and total concentration results in comparison to trypan blue based cell counting method. Thus, its demand along with fluorescent cell counter is increasing.
In this post, three types of commonly used fluorescent dye will be introduced.
Membrane-impermeant
Propidium Iodide (PI)
Definition:
PI is a fluorescent dye used to stain cells for cell counting. It is a membrane-impermeable dye which only stains the dead cells.
Mechanism:
Similar to TB, PI only stains the dead (non-viable) cells by entering through the damaged cell membrane and distinguishes the dead and live cells. PI binds to DNA inside the cell and emits a red fluorescent signal.
DAPI
Definition:
DAPI is another fluorescent dye staining DNA inside cells. Similar to PI, DAPI is membrane-impermeable dye so that it only penetrates through dead cells.
Mechanism:
As cell membrane of dying or dead cell breaks apart, DAPI enters into the cell and stains DNA. DAPI then emits blue fluorescent signal after binding. Thus, it is useful for cell quantification and obtaining viability of cell.
Membrane permeant
Acridine Orange (AO)
Definition:
AO is another type of fluorescent dye that stains cells to quantify cells in samples.
Mechanism:
The difference between TB or DAPI and AO is that AO is a membrane-permeant dye so it stains both live and dead cells regardless of the existence of cell membrane. AO emits two colors – green and red – depending on where it is bound to.
> When bound to DNA – emits green fluorescent signal
> When bound to RNA – emits red fluorescent signal
NanoEntek Fluorescent Cell Counter
ADAM MC2 / CellT
Analysis time | < 25 seconds |
Dye | PI, AO |
Cell type | Cell line, PBMCs, adipose stem cell, primary cells etc. |
Measuring range | 5x10E4 to 2x10E7 cell/mL |
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