Peter Kierulf wrote: >We as many others have been using trypane blue staining to account for >number og dead cells. >We have tried in vain to find a basic reference that gives information as >to why the cells become trypane blue positive when they are dead. >Can anyone help out?? Trypan blue staining works pretty much the same way propidium staining for dead cells works; it depends on loss of membrane integrity in dead cells. Acid dyes, such as trypan blue, and dyes with double positive charges, such as propidium, are normally excluded by intact cell membranes. When there is sufficient damage to the membrane, these dyes can enter the cell. Acid dyes such as trypan blue bind to intracellular protein; dyes such as propidium bind to nucleic acids. In general, cells in a culture which are permeable to trypan blue or propidium are dead; however, cells which are reproductively dead (e.g., after lethal irradiation or chemotherapy aimed at nucleic acids) may retain membrane integrity for days. Also, one can make cells transiently permeable to trypan blue or propidium with lysolecithin, heat or cold shock, electroporation, etc. For further information and references, see pp. 247 and 307 of the 3rd Edition of Practical Flow Cytometry. -Howard
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