Time-resolved flow cytometry for the measurement of lanthanide chelate fluorescence: I. Concept and theoretical evaluation.

The concept of a flow cytometer suited for the time-resolved measurement of lanthanide chelate luminescence with a decay time on the order of 10 microseconds to 2 ms is presented and evaluated. The instrument proposed encompasses a continuous-wave laser for fluorescence excitation and an optical switch for the elimination of cellular autofluorescence decaying within 1 ns to 1 microseconds during the luminescence detection period. The slowly decaying fluorescence is to be quantified by a photon-counting system, whereas light scatter and prompt fluorescence parameters are acquired by a conventional detection system. The detection limit of the method, in terms of the smallest detectable number of fluorescing chelates per cell, is examined. It was found to be nearly 30,000 complexes of a europium chelate with a decay time of 1.6 ms and a quantum efficiency of 17%, independent of fast decaying cellular autofluorescence or prompt dye emission intensity. The probability of cells passing through the instrument without being detected while the laser beam is turned off was estimated, and the implications for cell throughput and sorting performance of the instrument were assessed. At typical fluorescence detection intervals of 500 microseconds to 1 ms, cell flow rates of 100-200 particles per second lead to detection probabilities of more than 90% and sorting purities comparable to those found in conventional fluorescence-activated cell sorting.

Time-resolved flow cytometry for the measurement of lanthanide chelate fluorescence: II. Instrument design and experimental results.

A time-resolved flow cytometer capable of measuring a luminescence with a decay time in the range of 10 microseconds to 2 ms, typical for some lanthanide chelates, is presented. The instrument permits acquisition of conventional light scatter and prompt fluorescence signals as well as detection of slowly decaying luminescence by a photon counting unit for a selectable time period of 1 microsecond to 1 ms. During photon counting, the laser beam is turned off by an acoustooptic deflector. The design of a flow chamber with an average geometrical light collection efficiency of 35% over a distance of 1.7 mm is presented and analyzed by ray tracing. A pulse processing system featuring digital integration of the conventional signals and a transputer system for the acquisition and the transfer of the measured parameter values to a host computer is described. Instrument function is verified with lyophilized human lymphocytes stained for the CD8 antigen with dye-loaded liposomes. Quantitation of cell-associated europium chelate fluorescence, displaying a decay time of 1.6 ms, is demonstrated. Elimination of fast decaying background emission generated by DNA-associated ethidium bromide is shown. The background generated by instrument components in the time-gated measurement channel is characterized, and measures for its complete elimination are discussed.

Immunization-induced decrease of the CD4+:CD8+ ratio in cats experimentally infected with feline immunodeficiency virus.

In a previous experiment a group of 15 specified pathogen free (SPF) cats were experimentally infected with a Swiss isolate of feline immunodeficiency virus (FIV). A group of 15 SPF cats served as FIV negative controls. Nine cats of each group were vaccinated with a recombinant feline leukemia virus (FeLV) vaccine, six cats in each group with a placebo vaccine. All vaccinated cats developed high antibody titers to FeLV and were protected against subsequent FeLV challenge infection. In both control groups five of six cats became persistently infected with FeLV. Unexpectedly, the primary immune response to the vaccine antigen was significantly higher in the FIV positive group than in the FIV negative. The secondary response was stronger in the FIV negative cats. The goal of the present investigation was to further study the immune response in these 30 cats. They were immunized twice with the synthetic peptide L-tyrosine-L-glutamic acid-poly(DL-alanine)-poly(L-lysine) (TGAL) 21 days apart. Blood samples were collected on four occasions during the immunization process. They were tested for antibodies to TGAL, complete blood cell counts and CD4+, CD8+ and pan-T-lymphocyte counts. The following observations were made: (1) in contrast to the FeLV vaccine experiment, the primary immune response to TGAL was not significantly stronger in the FIV positive cats when tested by enzyme-linked immunosorbent assay (2). The absolute size of the CD4+ lymphocyte population was distinctly smaller in the FIV positive than in the FIV negative cats. The lowest CD4+ values were found in the dually FIV/FeLV infected cats. (3) A population of CD8+ lymphocytes was identified that was characterized by a distinctly weaker fluorescence. The size of this population increased in FIV positive and decreased in FIV negative cats during the TGAL immunization experiment. (4) The CD4+:CD8+ ratio increased in FIV negative cats during TGAL immunization from 1.9 to 2.3. In contrast, in FIV positive animals the CD4+:CD8+ ratio decreased significantly from 1.9 to 1.3 during the same period. From these and earlier data it was concluded that in short-term FIV infection the immune response to T-cell dependent antigens may be increased over that of the controls. Immune suppression develops gradually with duration of the infection. The significant drop of the CD4+:CD8+ ratio over a 5 week immunization period suggests that antigenic stimulation may accelerate the development of immune suppression in FIV positive cats. If this is a general feature, FIV infection may provide a particularly interesting model for studying the pathogenesis of AIDS.

Neurite outgrowth on immobilized axonin-1 is mediated by a heterophilic interaction with L1(G4).

Axonin-1 is an axon-associated cell adhesion molecule with dualistic expression, one form being glycophosphatidylinositol-anchored to the axonal membrane, the other secreted from axons in a soluble form. When presented as a substratum for neuronal cultures it strongly promotes neurite outgrowth from chicken embryonic dorsal root ganglia neurons. In this study, the axon-associated cell adhesion molecule G4, which is identical with Ng-CAM and 8D9, and homologous or closely related to L1 of the mouse and NILE of the rat, was investigated with respect to a receptor function for axonin-1. Using fluorescent microspheres with covalently coupled axonin-1 or L1(G4) at their surface we showed that these proteins bind to each other. Within the sensitivity of this microsphere assay, no interaction of axonin-1 with itself could be detected. Axonin-1-coated microspheres also bound to the neurites of cultured dorsal root ganglia neurons. This interaction was exclusively mediated by L1(G4), as indicated by complete binding suppression by monovalent anti-L1(G4) antibodies. The interaction between neuritic L1(G4) and immobilized axonin-1 was found to mediate the promotion of neurite growth on axonin-1, as evidenced by the virtually complete arrest of neurite outgrowth in the presence of anti-L1(G4) antibodies. Convincing evidence has recently been presented that neurite growth on L1(8D9) is mediated by the homophilic binding of neuritic L1(G4) (1989. Neuron. 2: 1597-1603). Thus, both L1(G4)- and axonin-1-expressing axons may serve as "substrate pathways" for the guidance of following axons expressing L1(G4) into their target area. Conceivably, differences in the concentration of axonin-1 and L1(G4), and/or modulatory influences on their specific binding parameters in leading pathways and following axons could represent elements in the control of axonal pathway selection.

Flow cytometric analysis of intracellular pH in cultured opossum kidney (OK) cells.

Suspensions of OK cells (a continuous renal epithelial cell line originating from the opossum kidney) were examined by flow cytometry. Three parameters were evaluated simultaneously; cell integrity as assayed by propidium iodide fluorescence, cell size as measured by time-of-flight, and intracellular pH as measured by fluorescence of 2',7'-bis-(2-carboxyethyl)-5,6 carboxyfluorescein (BCECF). The suspension was shown to be composed of both intact singlets and doublets of cells, and no difference was noted in the behavior of these two populations with respect to the resting intracellular pH, or of the response of intracellular BCECF to changes in pH. Evidence suggests that using NH4 prepulses to create an acid load broadens the intracellular pH distribution. The population of OK cells demonstrates a recovery from this acid load which is very homogeneous with respect to its sensitivity to Na+ removal of EIPA (ethylisopropyl-amiloride), suggesting that virtually all cells utilize Na+/H+ exchange for this recovery. The data also suggest heterogeneity in the cellular pH recovery from an acid load with respect to the observed rates of Na+/H+ exchange. Despite this heterogeneity, the Na+/H+ exchanger is observed to focus the resting intracellular pH of the population to approximately pH 7.4-7.5. The response of the population to PTH suggests that the majority of cells respond to the hormone, and that the total Na+/H+ exchange in individual cells is only partially inhibited even in the presence of saturating PTH concentrations.