Trypan blue, also known as Trypan Blue, has a molecular formula of C₃₄H₂₄N₆Na₄O₁₄S₄, a molecular weight of 960.81 and CAS Number 72‑57‑1. It is a cell‑viability dye widely used to assess cell‑membrane integrity and cell survival rate, and represents one of the most common staining methods for dead‑cell identification in tissue and cell culture. The intact cell‑membrane of healthy living cells can exclude trypan blue, so these cells will not turn blue. In contrast, cells that have lost viability or possess damaged cell‑membranes are stained blue by trypan blue. Loss of membrane integrity is generally regarded as an indicator of cell death.
Since trypan blue can be phagocytosed by macrophages, it can serve as a vital stain for macrophages. Apoptotic bodies also exhibit trypan‑blue exclusion. After trypan‑blue staining, cell‑viability can be quantified relatively accurately either by direct microscopic counting or by counting cells from captured microscopic images. The staining procedure is simple and only requires 3‑5 minutes.
Instructions for Use (For Reference):
a. Cell collection
Adherent cells should first be digested with trypsin. Suspension cells can be collected directly. Harvested cells are centrifuged at 1000‑2000 rpm for 3 min, the supernatant is discarded, and a single‑cell suspension is prepared with appropriate dilution.
b. Trypan‑blue staining
Resuspend the cell pellet in 0.2 % trypan‑blue solution and incubate for 3 min. Three minutes is sufficient for staining; longer incubation is permissible but should not exceed 10 minutes.
c. Cell counting
Aspirate a small volume of stained‑cell suspension and count the cells using a hemocytometer. Dead cells appear blue, swollen and dull; live cells remain unstained, retain their normal morphology and are bright. For reliable quantitative results, count at least 500 cells per sample.
Cell viability = (Total cell count − Blue‑stained cell count) / Total cell count × 100 %
Precautions:
1. A small amount of granular precipitate may form during long‑term storage. Place the product in a 37 °C water bath for approximately 10 minutes to fully dissolve the precipitate. The reagent may be used normally once all precipitate has dissolved.
2. Trypan blue is toxic to humans. Exercise extreme caution during handling and take adequate protective measures to avoid skin contact or inhalation.
3. For scientific‑research‑use‑only by qualified personnel. Not for clinical diagnosis or therapy, and not for food‑ or pharmaceutical‑related applications.
4. Wear a laboratory coat and disposable gloves throughout the procedure for your safety and health.
