Biomedical subjects
Publications and source records attributed to S E Slezak.
Building upon earlier studies with fluorescent probes, the authors describe a new cell tracking compound, PKH95, with a radioactive signal, which has been developed specifically for high-sensitivity cell tracking and biodistribution studies.
Studies of in vivo cell migration using cell markers such as 51Cr, 111In, FITC, or XRITC have been limited to short time periods due to the elution, toxicity, or rapid loss of label detectability. We have labeled sheep lymphocytes in vitro with PKH-2, a new fluorescent cell membrane label, and, after their intravenous injection back into donor sheep, have been able to detect them in efferent lymph, using flow cytometry, for longer than 38 days. The PKH-2-labeled lymphocytes migrated with similar kinetics, efficiency, and tissue specificity as lymphocytes labeled with cell markers used previously. PKH-2-labeled cells mediated graft versus host reactions indistinguishable from those mediated by unlabeled cells, and cell surface antigens were equally detectable on the surface of labeled and unlabeled lymphocytes. According to the slow, consistent loss of fluorescence intensity of the labeled cells in vivo, we predict that labeled lymphocytes could remain detectable by flow cytometry for greater than 7 weeks with the labeling protocol used in these experiments.
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We report a new technology for in vivo tracking of hematopoietic cells, using fluorescent lipophilic probes. Because the probe is irreversibly bound in the lipids of the cell membrane; substantial numbers of dye molecules can be incorporated per cell and thus substantial signal to noise can be achieved. Although this technology can be used for all hematopoietic cells, these first findings are reported on red blood cells (RBCs) owing to the importance of the membrane to RBC function and integrity. We demonstrated that labeling 10% of the RBCs of a rabbit and reinjecting them into the animal makes possible the tracking of these cells at various times after injection. Furthermore, the labeling appears not to affect in vivo cell lifetime or cellular volume changes in response to hypotonic shock. The single cell fluorescence intensity of the labeled RBCs remains relatively constant for 60 days, and an immune response appears not to be generated against labeled cells. That labeled RBCs have lifetime kinetics in vivo, as shown in other studies, indicates that the membranes are functioning normally and are unaltered by the labeling technology. The technology we present is also applicable to white blood cells, bone marrow, and platelets.
Binding fluorescent or radioactive reporter molecules to the lipid bilayer of cell membranes allows cell growth and trafficking to be monitored in vivo.
Determination of target cell lysis by cytolytic effectors has typically been achieved by two methods: the release of various markers from the cell, as in 51chromium release assays and the uptake of markers into the cell, as in trypan blue uptake in single cell/conjugate binding assays. Problems associated with these assays might include: (1) poor uptake, (2) nonspecific release, (3) poor statistics, (4) length of assays, or (5) subjectivity. These difficulties prompted the development of a new sensitive flow cytometric assay employing two fluorochromes. PKH-1, a fluorochrome which fluoresces in the green, binds to the cytoplasmic membrane and does not leak or transfer, is used to identify the target cell population. Propidium iodide fluoresces in the red and is used to detect non-viable cells. Use of these two fluorochromes and two parameter analysis allows for identification of four subpopulations in the sample: live effectors, dead effectors, live targets and dead targets. By enumeration of these subpopulations the following information can be calculated: (1) the percent target lysis, (2) effector-to-target cell ratios, (3) viability of the effector cells at the termination of the assay, and (4) viable effector to target cell ratios. The results show that PKH-1 labeling of target cells had no effect on effector-target cell interactions. Excellent correlation was found between this method and the chromium assay, however, due to earlier detection of the lytic event, this method provides a distinct time advantage over current methods.
Flow cytometric analysis of human peripheral blood leukocytes has typically been achieved by staining multiple aliquots of the same sample with fluorescent reagents specific for cell subsets of interest. Spectrally discrete fluorochrome tags have been developed for applications in which identification of multiple subsets (e.g., T and B cells) or of subsets not uniquely identified by a single reagent (e.g., activated T cells) requires use of multiple reagents per aliquot. Extension of this approach to more than two reagents per aliquot has led to multicolor methods requiring dual laser excitation and complex instrumentation. We describe an alternative two-color method using commercially available reagents that allows simultaneous identification of five discrete immune cell subsets using only a single excitation source. The technique uses dilution of commercial fluorochrome-labeled reagents with competing unlabeled reagents to selectively produce discrete fluorescence intensity profiles for cell subsets that would otherwise display overlapping or indistinguishable profiles when stained with reagents bearing the same fluorochrome. For example, the fluorescence intensity of phycoerythrin-labeled helper T (Th) cells can be adjusted to be distinct from that of phycoerythrin-labeled suppressor T (Ts) cells. Extending this technique to two colors, we have used a combination of seven different monoclonal antibodies to simultaneously quantify Th, Ts, B cells, natural killer cells, and monocytes in a single aliquot. An additional advantage of this approach is the ability to more accurately quantify "null" cells. Adjustment of fluorescence intensity profiles of different cell subsets by this method is applicable to flow cytometric analysis of a wide variety of cell types. The technique significantly extends the analytical capacity of flow cytometry without significantly increasing the complexity of the instrumentation required.