Trypan blue staining is a long-standing and widely used method to identify dead cells. Only cells with intact membranes can effectively exclude the dye, so dead cells with compromised membranes become stained. Trypan blue staining is the method employed by the Countess family of automated cell counters, and this 0.4% trypan blue solution is the correct concentration for use on these instruments.
Note: Precipitates are quite common in trypan blue, and typically increase with storage time and freezing. The three approaches below may help minimize trypan blue precipitate in your cell counting samples:
1. Sterile filtration of trypan blue using 0.3-micron filter
2. Centrifugation or passive settling (avoid mixing or vortexing trypan stock solutions)
3. Gentle warming of the 1-mL trypan solution to 37°C for ten minutes
