PubMed · 6864941
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D W Ross. WBC differential counts.. https://pubmed.ncbi.nlm.nih.gov/6864941/
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BACKGROUND: As immunofluorescence experiments become more complex, the demand for new dyes with different properties increases. Fluorescent dyes with large Stoke's shifts that are very bright and have low background binding to cells are especially desirable. We report on the properties of the resonance energy tandems of phycoerythrin and allophycocyanin (PE-APC). PE-APC is the original fluorescence resonance energy tandem dye described in the literature, but it has not been utilized because of the difficulty of synthesizing and preparing a consistent product. METHODS: PE-APC complexes comprising different ratios of the two phycobiliproteins conjugated to streptavidin were synthesized using standard protein-protein conjugation chemistry. The PE-APC streptavidins were evaluated for flow cytometric analysis. They were compared directly to Cy5PE conjugates because Cy5PE is the fluorophore that is spectrally most like the PE-APC. RESULTS: PE-APC complexes showed the expected fluorescence spectral properties of a tandem: excitation was excellent at 488 nm (and best at the PE excitation maximum) and emission was greatest at the APC emission maximum at about 660 nm. The efficiency of transfer of energy from PE to APC was about 90%. CONCLUSION: PE-APC can be considered an excellent substitute for Cy5PE. Compared with Cy5PE, PE-APC has similar brightness (in staining experiments), slightly greater compensation requirements with PE but much lower compensation with Cy5.5PE or Cy5.5PerCP, and lower nonspecific background binding. PE-APC is a useful alternative to Cy5PE, especially in applications in which the use of Cy5 is impractical. Cytometry 44:24-29, 2001. Published 2001 Wiley-Liss, Inc.
BACKGROUND: Conventional flow cytometry does not allow the rapid analysis of multiple samples. This has limited its uses in drug discovery, for which the standard for throughput is 100,000 samples per day. METHODS: We describe a simple method in which commercial peristaltic tubing is connected from a commercial autosampler to a flow cytometer. The samples are delivered via a peristaltic pump from source wells in a multiwell plate. The samples are separated by air bubbles. RESULTS: Throughput rates approach the limit of the autosampler (up to 100 wells per minute). Using optimal tubing and flow rates, particles remain within appropriate light scatter and fluorescence gates. The carryover between wells is typically less than 5% without and 1% with a wash step. The volumes of sample delivered are in the microliter scale. The approach has been validated with instruments from three manufacturers. CONCLUSIONS: Flow cytometry has potential throughput of 100,000 samples or more per day starting with the method described. The method is currently best suited to end-point assays. However, combined with high-speed sorting and single- cell assays, the number of assays could approach 1 billion per day.
BACKGROUND: Apart from the work of Lampariello and colleagues (Cytometry 15:294-301, 1994; Cytometry 32:241-254, 1998), very little analytical work has been carried out for analysis of immunofluorescence distributions containing an overlapping mixture of labeled and unlabeled cells. The methods developed tend to rely on fitting theoretical distributions to the relevant populations. However, the method described here attempts to produce an analytical solution. METHODS: A new method for immunofluorescence histogram analysis is presented. It uses cumulative frequency distribution subtraction of the test sample from the control to predict the mean of a labeled cell component embedded within a histogram containing unlabeled cells. Ratio analysis of means (RAM) was then carried out to calculate the labeled fraction. The results were submitted to Kolmogorov-Smirnov analysis and Student's t-test for validation at a given level of probability. RESULTS: The method was developed with a data set exhibiting a small "positive" shoulder, which was predicted to contain a labeled fraction comprising 8.0% of the total at the 99% confidence limit. It was then tested with data analyzed and published previously where the Johnson Su family of distributions was used in curve fitting. CONCLUSIONS: There was good agreement between the known and predicted proportions of labeled cells. However, the method is dependent on the symmetry of the distributions. Some minor systematic errors were encountered due, in part, to skewed experimental distributions.