How to Achieve Precise Cell Counting with AO/PI Staining?
| October 20, 2022
Cell concentration and viability detection is one of the most basic cell culture processes, and cell staining is required to perform cell counting and viability assays. The commonly used stains include trypan blue and acridine orange/propidium iodide (AO/PI).
Trypan blue is a stain commonly used to distinguish live cells from dead cells. Since the membrane of live cells is selectively permeable and can block trypan blue, live cells will not be stained. The membrane of dead cells is more permeable, so dead cells can be stained blue by trypan blue. If trypan blue staining is adopted in the experiment, the viability and concentration of the cell sample can be quickly calculated by observing and counting the stained and unstained cells under the microscope.
For more accurate cell-counting, trypan blue is not an ideal method. Since it usually stains the debris of primary cells and residual red blood cells in PBMCs. Besides, there is no standard for the preparation of dye solution, so that different solution may lead to different counting results. In addition, trypan blue is toxic to cultured cells, and it may destroy the cells if staining for a long time.
However, in practical applications, the actual results will be different due to the different cell types, staining conditions and parameters of detection instruments, in that case, it is hard to guarantee stable staining results.
The fluorescent dye AO can penetrate the cell membrane and stain DNA and RNA, respectively, producing green fluorescence(DNA) and orange fluorescence(RNA).
The fluorescent dye PI is a nuclear staining reagent that can stain DNA, which is commonly used for the detection of apoptosis. It is an analog of ethidium bromide, and emits red fluorescence after embedding itself in double-stranded DNA. PI cannot pass through the living cell membrane, but can only pass through the broken cell membrane to stain the nucleus.
Since debris, impurities, or mammalian red blood cells do not have a nucleus, they are not stained with AO/PI in practical applications. Therefore, the counting based on AO/PI staining is more accurate than that based on trypan blue staining.
Moreover, AO/PI staining has low toxicity toward cultured cells. Hence, it is widely used in primary cell counting, cell therapy, biological products, etc.
Protocol of AO/PI rapid staining
A variety of prepared dye solutions are available on the market. When cell samples are incubated in staining solution at a final concentration of 2-25 μg/mL for 5–30 min, they may emit bright nuclear staining fluorescence. However, due to the different cell types, staining conditions and parameters of detection instruments in practical applications, the actual results will be different, so it is hard to guarantee stable staining results.
To obtain stable staining results, cell staining and counting can be performed as follows:
1.Prepare premixed solution of AO and PI (10μg/mL AO, 50μg/mL PI);
2.Pipette 10 μL of cell sample into an EP tube, add 10 μL of premixed AO/PI solution and mix well, then incubate for 5 min (final concentration: PI, 25 μg/mL; AO, 5 μg/mL);
3.Gently mix the solution, then pipette 10 μL of stained cells into the cell counting plate for analysis.
RWD Automated Cell Counter is recommended. This device can accurately and rapidly distinguish living cells from dead cells stained with AO/PI dual fluorescence by one-click counting.
By adopting AO/PI fluorescent cell counting method, RWD Automated Cell Counter can deliver accurate and stable cell staining counting. It is widely used in multiple application scenarios such as cell count analysis, cell viability analysis, cell size analysis, and cell transfection efficiency (GFP, RFP) detection, helping researchers to efficiently and accurately analyze cell concentration, viability, size and fluorescence intensity.