Trypan blue, also known as Trypan blue stain, has the molecular formula C₃₄H₂₄N₆Na₄O₁₄S₄, a molecular weight of 960.81 and CAS No. 72-57-1. It is an anionic dye. Trypan blue cannot penetrate the intact cell membrane of viable cells, so living cells remain unstained, which is known as the dye exclusion effect. In contrast, the cell membrane permeability of dead cells increases, allowing the dye to enter the cells and stain dead cells blue. It is the most commonly used method for cell viability detection. Generally speaking, the loss of cell membrane integrity indicates cell death, which is opposite to the staining principle of neutral red. Therefore, trypan blue staining enables simple and rapid differentiation between live and dead cells, and it is one of the most widely used staining methods for dead cell identification in tissue and cell culture.
Trypan Blue Solution (0.4%) is commonly used to detect cell membrane integrity and cell viability. Live cells are not stained blue, while dead cells turn pale blue. Since trypan blue can be phagocytosed by macrophages, it can serve as a vital stain for macrophages. After trypan blue staining, cell viability can be accurately quantified via direct microscopic counting or counting based on microscopic images, with 0.4% being the most frequently used working concentration. This reagent is only for scientific research purposes and is not applicable to clinical diagnosis or other uses.
Usage Method:
1. Cell collection
For adherent cells, digest cells with trypsin-EDTA solution before collection; for suspension cells, collect directly. Centrifuge the harvested cells at 1000~2000 g for 1 minute, discard the supernatant, and resuspend the cell pellet in 1 mL PBS.
2. Trypan blue staining
Transfer 100 μL cell suspension into a 1.5 mL or 0.5 mL centrifuge tube, add 100 μL Trypan Blue Solution (0.4%) and mix gently. Let stand for 3 minutes for staining. The staining time can be properly extended, but shall not exceed 10 minutes.
3. Cell counting
Take the stained cell mixture and count cells with a hemocytometer. For accurate quantification, count no less than 500 cells per sample, and record the number of blue-stained cells and total cells.
4. Cell viability calculation formula
Cell Viability = (Total Cell Number − Blue Cell Number) / Total Cell Number × 100%
Precautions:
1. Trypan Blue Solution (0.4%) is slightly irritating to the human body. Please take proper protective measures during operation.
2. Note that apoptotic bodies may occasionally show trypan blue dye exclusion.
3. For your personal safety and health, please wear lab coats and disposable gloves when handling.
Disclaimer: Our company shall not be liable for any accidents caused by improper use of this product.
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