PubMed · 8531764
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J Sheen. 1995. Methods for mesophyll and bundle sheath cell separation.. https://doi.org/10.1016/s0091-679x(08)61462-4
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Cytotoxic T lymphocytes (CTLs) play an important role in controlling viral infections and certain tumours, but characterising specific CTL responses has always been technically limited. Fluorogenic 'tetramers' of major histocompatibility complex (MHC) class I complexes have been exploited recently to quantify the massive expansion of specific CTLs in human immunodeficiency virus (HIV) infection [1]. Here, we use MHC class I complex tetramers to isolate low-frequency antigen-specific CTLs directly from human peripheral blood, allowing the simultaneous phenotypic and functional characterisation and cloning of these CTLs. We synthesised a tetramer that specifically stained human leukocyte antigen (HLA)-A2. 1-restricted CTL clones recognising the influenza matrix protein peptide 58-66, matrix 58-66 [2]. This tetramer stained between 1 in 1,500 and 1 in 58,000 peripheral blood mononuclear cells (PBMCs) from HLA-A2.1+ individuals. The surface phenotype of these cells could be analysed by fluorescence-activated cell sorting (FACS), and the cells could be directly sorted into enzyme-linked immunospot (ELISpot) plates, where they released interferon-gamma (IFN-gamma) within 1 day of antigen exposure. The same population was cloned by FACS, and the specificity of several expanded clones was confirmed. Cloning was greatly simplified and accelerated compared with standard protocols, and was highly efficient. We also used tetramer-based sorting to enrich melanoma-specific CTLs derived from a tumour-infiltrated lymph node. Direct cloning of specific CTLs from peripheral blood can provide important information about immunological memory, CTL responses against tumour antigens and CTL proliferation and function, and opens up new possibilities for generating CTLs for adoptive immunotherapy.
White blood cells are isolated from whole blood in silicon-glass 4.5-microliter microchips containing a series of 3.5-micron feature-sized 'weir-type' filters, formed by an etched silicon dam spanning the flow chamber. Genomic DNA targets, e.g., dystrophin gene, can be directly amplified using the polymerase chain reaction (PCR) from the white cells isolated on the filters. This dual function microchip provides a means to simplify nucleic acid analyses by integrating in a single device two key steps in the analytical procedure, namely, cell isolation and PCR.
The DNA content and light scatter from individual Escherichia coli cells were measured in two flow cytometers with different configurations. The DNA content could be measured with similar resolution either in an Argus flow cytometer equipped with a mercury lamp, or in a FACStar flow cytometer with two argon lasers as light sources. In contrast, light scatter measurements appeared to be a good measure of cell mass only in the Argus instrument. Three DNA stains were compared:, DAPI, Hoechst 33258, and mithramycin A together with ethidium bromide. All three stains yielded DNA histograms of similar quality in both flow cytometers. Optimal results required that the stain and cell concentrations were kept similar, that a fixed rate of sample introduction was used, and that a period of equilibration was allowed during running of each sample. The results demonstrate that conventional, laser-based flow cytometers may be used for high-resolution measurements of bacterial DNA content, thereby making flow cytometry available to an increased number of research groups working with prokaryotes.