| 主权项 |
1. A method for analyzing detergent-liberated nuclei and/or chromatin debris for nuclei-associated epitopes, the method comprising:
simultaneously contacting a sample containing eukaryotic cells with a reagent solution comprising one or more cell lysis agents, a fluorescent nucleic acid dye (NAD), and one or more fluorescent nuclei-associated epitope-specific antibodies or ligands (NAESA/L) that specifically bind to one or more nuclei-associated epitopes of interest to thereby form a test suspension, wherein the NAD and each of the one or more NAESA/L have distinct fluorescent emission spectra, and said simultaneous contacting with the reagent solution digests eukaryotic cell cytoplasmic membranes but not nuclear membranes, aggregates metaphase chromosomes of a single cell into a single bundle of chromosomes, labels chromatin with the NAD, and labels the one or more nuclei-associated epitopes with the one or more NAESA/L; exciting the NAD and one or more NAESA/L in the test suspension with light of an appropriate excitation wavelength; detecting fluorescent emission and light scatter produced by the nuclei and/or chromatin debris in the test suspension and counting one or more of the following events: the number of nuclei, the number of nuclei positively labeled by the one or more NAESA/L, the number of chromatin debris, the number of chromatin debris positively labeled by the one or more NAESA/L, the number of nuclei in G1, S, and G2/M phases of the cell cycle, and the number of polyploid nuclei; and determining one or more of the following measurements: the frequency of nuclei positively labeled by the one or more NAESA/L relative to total nuclei, the frequency of chromatin debris positively labeled by the one or more NAESA/L relative to total chromatin debris and/or total nuclei, the proportion of nuclei in G1, S, and G2/M phases of the cell cycle, the proportion of polyploid nuclei, and mean and/or median fluorescence by the one or more NAESA/L. |
