A device capable of separating biological cells (suspended in a conducting medium) according to volume has been developed. Cell volume is measured in a Coulter aperture, and the cells are subsequently isolated in droplets of the medium which are charged according to the sensed volume. The charged droplets then enter an electrostatic field and are deflected into a collection vessel. Mixtures of mouse and human erythrocytes and a large volume component of mouse lymphoma cells were separated successfully. In tests with Chinese hamster ovary cells essentially all cells survived separation and grew at their normal rate.

An instrument is described in which two solutions can be homogeneously mixed within several microseconds. The liquids flow separately through two coaxial capillaries with conical tips and then simultaneously around a sphere (50–100 μ in diameter) which has been positioned close to the end of the outer tip. The liquids flow with velocities of ∼100 m/s through the small passages (∼5 μ wide) separating the sphere and the wall of the outer capillary and mix in the turbulent liquid flow behind the sphere. The mixed liquids are then ejected as a narrow liquid jet for observation. Design characteristics and construction techniques are presented along with a discussion of the properties of the turbulent flow field and estimates of the expected practically realizable mixing times. The experimentally determined speed of mixing indicates that we have nearly achieved the proposed lower limits of the mixing time.

A new flow-system instrument for quantitative analysis and sorting of microscopic particles, particularly biological cells, based on multiple measurements of physical and biochemical properties has been developed. Cells stained with fluorescent dyes in liquid suspension enter a unique flow chamber where electrical and optical sensors measure cell volume, single- or two-color fluorescence, and light scatter, and emerge in a liquid jet that is broken into uniform droplets. Sensor signals are electronically processed several ways for optimum cell discrimination and are displayed as pulse-amplitude distributions using a pulse-height analyzer. Processed signals trigger cell sorting according to preselected parametric criteria. Sorting is accomplished by electrically charging droplets containing the cells and electrostatically deflecting them into collection vessels. This instrument is described in detail with illustrative examples of experiments using polystyrene fluorescent microspheres, cultured human cells, and human leukocytes.

Two monoclonal antibodies (OKT27 and OKT27b) have been produced that react with distinct epitopes of a 95-kDa peptide. The T27 antigen is widely distributed, being expressed on B lymphocytes, monocytes, and adult Tleukemic cells but not on polymorphonuclear leukocytes or platelets. There was a low level of T27 expression on resting T cells that increased on T-cell activation. In preliminary studies, the OKT27b antibody coprecipitated a 55-kDa peptide, as well as the 95-kDa peptide, from the radiolabeled cells of the HuT 102B2 cell line. Preclearance with anti-Tac, a monoclonal antibody to the 55-kDa peptide of the multichain interleukin 2 receptor, removed the 55-kDa but not the 95-kDa peptide from subsequent OKT27b immunoprecipitates of HuT 102B2 extracts, suggesting the possibility that the T27 peptide was associated with the Tac peptide. However, the precipitation of the p55 Tac peptide by OKT27b was quite inconsistent. Thus, additional information was sought using a flow cytometric energy transfer technique to provide a physical estimation of the proximity between the Tac and the T27 peptides. The flow cytometric version of the fluorescence resonance energy transfer technique permits the determination of inter-and intramolecular distances at 2-to 10-nm levels on a cell-by-cell basis. Using this approach, there was a mean energy transfer of 7.3% with HuT 102B2 cells when fluorescein isothiocyanate anti-Tac served as the donor and tetramethylrhodamine isothiocyanate OKT27 served as the acceptor. In contrast, there was no energy transfer in comparable studies observed when fluorescein anti-Tac and rhodamine anti-transferrin receptor antibodies were used. These observations support the conclusion that there is a close nonrandom proximity in HuT 102B2 cells between the 95-kDa peptide identified by the OKT27 monoclonal antibody and the p55 Tac peptide of the multichain interleukin 2 receptor.T lymphocytes stimulated with antigen or mitogen produce interleukin 2 (IL-2) (1, 2). T-cell growth is dependent on the interaction of IL-2 with high-affinity IL-2 receptors that are not present on resting cells but are induced and expressed on T cells after activation (3-5). There are at least two classes of IL-2 receptors that differ markedly in their affinities for IL-2 (6). Both classes of receptors share the same Tac peptide (55 kDa) (4,5). To define the molecular basis for high-and low-affinity receptors and to determine the mechanism whereby IL-2 communicates a signal to the nucleus, we have investigated the possibility that the IL-2 receptor is a complex with multiple peptide chains in addition to the one identified by anti-Tac (7). Using crosslinking methodology, we have identified a non-Tac IL-2 binding peptide of 75 kDa and have proposed that the 75-kDa IL-2 binding peptide is associated with the 55-kDa Tac peptide to form the highaffinity IL-2 receptor complex (7). Recently, two additional monoclonal antibodies, OKT27 and OKT27b, were developed in an effort to identify distinct human T-cell activation antig...

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Flow cytometric energy transfer (FCET)measurements between labeled specific sites of cell surface elements (Szollosi et al., Cytometry, 5210-216, 1984) have been extended in a simplified form using a flow cytometer equipped with single excitation beam. This versatile and easily applicable method has several advantages over any nonflow cytometric (i.e., spectrofluorimetric) energy transfer measurements on cell surfaces: 1) The labeled ligands can be applied in excess, without washing, thereby enabling the investigation of relatively labile receptor-ligand complexes. 2) Contributions of signals from cell debris, from cell aggregates, or from nonviable cells can be avoided by gating the data collection on the light scatter signal.3) The heterogeneity of the cell population with respect to the proximity of the labeled binding sites can be studied. 4) In the cases of homologous ligands or of ligands binding to the same molecule but at different epitopes, the determination of fluorescence resonance energy transfer efficiency values can be carried out on a cell-by-cell basis, offering data on intramolecular conformational changes.This modified FCET method enabled us to demonstrate the uniform density of glycoproteins, specific for Con A binding, in the plasma membrane of normal and Gross virus leukemic mouse cells of different sizes. The utility of this procedure has also been demonstrated by using the mean fluorescence intensities of the distribution curves in the calculation of the fluorescence energy transfer efficiency.Key terms: Resonance energy transfer, concanavalin A, Gross virus leukemia, flow cytometry, flow microfluorimetry, receptor densityThe cytoplasmic cell surface membrane plays a major role in communication between the environment and the cell interior. The cell senses specific external stimuli through surface receptors. The information necessary for cell function can be communicated by the aggregation state of these receptors or by changes in the distribution state of receptors on the membrane.The proximity relationship of membrane receptors can be studied by measuring the efficiency of fluorescence resonance energy transfer (FRET) between donor and acceptor fluorophores conjugated to components in question (21). FRET measurements have been performed on cell surfaces using spectrofluorimetry (4,lB-201, microscopy (91, and flow cytometry (3,5,10,(13)(14)(15)24,25). The FRET efficiencies determined using spectrofluorimeters are values averaged over the entire homogeneous or heterogeneous population. The data obtained with spectrofluorimeters are highly perturbed by the amount of debris, dead cells, aggregates, etc. Furthermore the procedures used to eliminate excess labeled ligands also remove cells from the samples and present a n almost insurmountable problem when comparing fluorescence intensities of single-labeled samples with those of double-labeled samples. We have developed and recently published (5,24,25) a new method using a flow cytometer capable of dual-wavelength excitation to determine FRE...

Chicken erythrocytes orient when caught in the confluence of two sheath-liquid streams. Preliminary results indicate that this phenomena might be used to align cells for morphologic analysis in flow.

A new technique has been developed for producing microspheres uniform in volume and fluorescence or radioactive tracer. Very uniform spheres with selected diameters are produced by introducing one liquid into the center of a moving stream of a second liquid (of differing surface tension) and breaking the core stream into uniform droplets by the action of a piezoelectric transducer. Fluorescent spheres of 10 μ diam have been produced having a coefficient of variation in volume and fluorescence as low as 3.6%. This corresponds to a diameter variation of about 1.5%.