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Fluorescent viability assay using cyclic-substituted unsymmetrical cyanine dyes

  • US 5,534,416 A
  • Filed: 11/08/1993
  • Issued: 07/09/1996
  • Est. Priority Date: 04/13/1993
  • Status: Expired due to Term
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First Claim
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1. A method of analyzing cell viability, comprising:

  • a) combining a sample of cells with an aqueous dye solution comprising a fluorescent Dye I of the formula;

    ##STR16## where t=1-4 and each R1, which may be the same or different, is H, C1-6 alkyl;

    trifluoromethyl;

    halogen;

    or --OR8, --SR8 or --(NR8 R9) where R8 and R9, which can be the same or different, are independently H;

    C1-6 alkyl;

    or 1-2 alicyclic, heteroalicyclic, aromatic, or heteroaromatic rings having a total of 3-16 ring atoms wherein the heteroatoms are O, N or S;

    or R8 and R9 taken in combination are --(CH2)2 --L--(CH2)2 -- where L=a single bond or --CH2 --, --O--, or --NR10, where R10 is H or C1-6 alkyl;

    R2 is C1-6 alkyl;

    Z- is a biologically compatible counterion;

    X is O;

    S;

    Se;

    or NR15, where R15 is H or C1-6 alkyl;

    or CR16 R17, where R16 and R17, which may be the same or different, are independently H or C1-6 alkyl, or the carbons of R16 and R17 taken in combination complete a five or six membered saturated ring;

    n=0, 1, or 2;

    Q has the formula Q1 or Q2 ##STR17## wherein Y is --CR3 ═

    CR4 --;

    p and m=0 or 1, such that p+m=1;

    R5 is a C1-6 alkyl, C1-6 alkenyl, C1-6 polyalkenyl, C1-6 alkynyl or C1-6 polyalkynyl group;

    or R5 is an OMEGA;

    R3, R4, R6 and R7, which may be the same or different, are independently H;

    or a C1-6 alkyl, C1-6 alkenyl, C1-6 polyalkenyl, C1-6 alkynyl or C1-6 polyalkynyl group;

    or halogen;

    or --OR8, --SR8, --(NR8 R9), as defined previously;

    or --OSO2 R19 where R19 is C1-6 alkyl, or C1-6 perfluoroalkyl, or aryl;

    or an OMEGA;

    or R6 and R7, taken in combination are --(CH2)v -- where v=3 or 4, or R6 and R7 form a fused aromatic ring according to formula Q2;

    R11, R12, R13, and R14, which may be the same or different, are independently H;

    or a C1-6 alkyl, C1-6 alkenyl, C1-6 polyalkenyl, C1-6 alkynyl or C1-6 polyalkynyl group;

    or a halogen;

    or an OMEGA;

    or --OH, --OR8, --SR8, or --(NR8 R9);

    OMEGA is a saturated or unsaturated, substituted or unsubstituted, cyclic substituent that has a total of 2-16 ring carbon atoms in 1-2 alicyclic, heteroalicyclic, aromatic, or heteroaromatic rings, containing 1-4 heteroatoms wherein the heteroatoms are O, N or S, that is unsubstituted or optionally substituted one or more times, independently, by halogen, alkyl, perfluoroalkyl, amino, alkylamino, dialkylamino, alkoxy or carboxyalkyl, having 1-6 carbons, and that is attached as R3, R4, R5, R6, R7, R11, R12, R13, or R14 by a single bond;

    such that at least one of R3, R4, R5, R6, R7, R11, R12, R13, and R14 is an OMEGA, and, where more than one of R3, R4, R5, R6, R7, R11, R12, R13, and R14 is an OMEGA, each OMEGA is optionally the same or different;

    and a fluorescent Dye II that selectively stains either viable or non-viable cells, where Dye II and Dye I together intracellularly have a detectable fluorescent response in combination with cells in the sample that is different from a fluorescent response of Dye I alone, and where Dye I and Dye II are each present in an amount sufficient to give a detectable fluorescent response, resulting in a sample of stained cells;

    b) illuminating the sample of stained cells at a suitable absorption wavelength that results in one or more illuminated cells; and

    c) observing the illuminated cells with means for detecting a fluorescent response resulting from illumination.

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