Trypan Blue Staining Solution (0.4%) is commonly used to assess cell membrane integrity and cell viability. Live cells remain unstained, while dead cells are stained light blue. Trypan Blue can be phagocytosed by macrophages, making it suitable for use as an in vivo stain for macrophages. After staining, cell viability can be accurately quantified by direct counting under a microscope or by counting from microscopic images. The concentration of 0.4% is the most commonly used concentration.
Protocol
1. Cell Collection
If collecting adherent cells, digest the cells with trypsin-EDTA solution, then harvest them. For suspension cells, cells can be collected directly. Centrifuge the collected cells at 1000 rpm for 5 minutes, discard the supernatant, and resuspend the cell pellet in 1 mL of PBS.
2. Trypan Blue Staining
It is recommended to add 25 μL of Trypan Blue stock solution (0.4%) to 75 μL of the cell suspension and mix gently. The final concentration will be 0.1% (w/v). Depending on the sample and instrument, the final concentration may range from 0.04% to 0.2% (w/v). Stain at room temperature for 3 minutes (staining time can be appropriately extended, but should not exceed 10 minutes).
3. Cell Counting
Transfer a small amount of the stained cell suspension to a hemocytometer for counting. Under the microscope, dead cells appear blue, swollen, and dull, while live cells remain unstained and exhibit normal morphology with a glossy appearance. For accurate quantification, at least 500 cells per sample should be counted. Count both the blue-stained (dead) cells and the total number of cells. Cell viability is calculated using the following formula:
Cell Viability (%) = (Total number of cells – Number of blue cells) / Total number of cells × 100%
Notes
1. Trypan Blue is slightly toxic to humans. Please take proper precautions to avoid exposure.
2. Note that apoptotic bodies may occasionally exhibit Trypan Blue exclusion as well.
3. For your safety and health, please wear a lab coat and disposable gloves during handling.
Results Presentation
Figure 1
Figure 1. Viability test of K562 cells using Trypan Blue staining. Cells were collected by centrifugation (1000 rpm, 5 min), resuspended in PBS. Stain for 3 minutes using either 750 μL cell suspension + 250 μL Trypan Blue (4×) or 900 μL cell suspension + 100 μL Trypan Blue (10×). Blue-stained cells were counted as dead cells.
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