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Viability Assay Technique

IP.com Disclosure Number: IPCOM000092062D
Original Publication Date: 1968-Aug-01
Included in the Prior Art Database: 2005-Mar-05
Document File: 1 page(s) / 12K

Publishing Venue

IBM

Related People

Kamentsky, LA: AUTHOR [+2]

Abstract

This rapid and automatic technique for discriminating live from dead cells provides data from which percent viability can be calculated. A population of biological cells is suspended in a dye mixture comprising a dye selected from trypan blue, eosin and the like and fluorescien-diacetate. Live cells actively reject trypan blue, etc., and remain colorless. Dead cells accumulate the dye. The fluorescien-diacetate is enzymatically degraded by the live cells to fluorescien, which accumulates in the live cells. Fluorescein fluoresces under appropriate excitation. Dead cells do not accumulate sufficient levels of fluorescein to similarly fluoresce.

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Viability Assay Technique

This rapid and automatic technique for discriminating live from dead cells provides data from which percent viability can be calculated. A population of biological cells is suspended in a dye mixture comprising a dye selected from trypan blue, eosin and the like and fluorescien-diacetate. Live cells actively reject trypan blue, etc., and remain colorless. Dead cells accumulate the dye. The fluorescien-diacetate is enzymatically degraded by the live cells to fluorescien, which accumulates in the live cells. Fluorescein fluoresces under appropriate excitation. Dead cells do not accumulate sufficient levels of fluorescein to similarly fluoresce.

The suspension of blue-stained dead cells and fluorescent live cells is flowed past the objective lens of a rapid cell analyzer. The light source of the analyzer, after filtering, produces light which includes wavelengths which are absorbed by trypan blue and which cause fluorescein to fluoresce. Light emerging from the objective lens of the analyzer is split by a dichroic mirror which directs those wavelengths which trypan blue absorbs, but which are not included in the emission band of fluorescein, to a first multiplier tube. The remainder of the light which includes the emission band of fluorescein is filtered to remove all wavelengths originating from the filter source and is directed to a second photomultiplier tube. The dead cells create a lowering of the background intensity at the first phot...