ABSTRACT

A new flow cytometric method has been developed to rapidly determine sperm concentration and viability in semen from bulls and boars. Sperm viability was determined on the basis of staining with SYBR-14 and propidium iodide (PI), and this allowed detection of live (membrane-intact) sperm, dying (moribund) sperm, as well as dead cells. Fluorescent microspheres (beads) were used to determine sperm concentration. The use of SYBR-14 at 50 nM and PI at 12 micro M in combination with the FACSCount diluent in the counting tubes resulted in a uniform staining after 2.5-5 minutes at room temperature. Reagent staining was reproducible enough to allow subsequent semiautomated analysis of data using Attractors software. In experiment 1, this method was used to analyze semen from boars, rams, rats, rabbits, humans, and turkeys. In experiment 2, Attractors analysis was performed by the FACSCount AF flow cytometer, and sperm concentration determination with this system was compared with results obtained by a spectrophotometer and an electronic cell counter, which is routinely used by bull artificial insemination centers. When compared to microscopic counting in a hemocytometer, the FACSCount AF flow cytometer was two and four times more accurate than the spectrophotometer and the electronic cell counter, respectively. In addition, the FACSCount AF flow cytometer determined both sperm concentration (coefficient of variation 3.3%) and sperm viability (coefficient of variation 0.7%) with high precision.

No abstract

Antibody-directed targeting of vesicles to cells dramatically enhances polyethylene glycol-mediated fusion and microinjection. Sealed erythrocyte ghosts, containing fluorescent bovine serum albumin, were targeted to murine spleen and thymus cells, and to lymphocyte, monocyte, and fibroblast cell lines. In all cases, targeted cell populations showed substantial levels of microinjection, whereas populations treated with the fusogen in the absence of targeting were not significantly microinjected. To achieve attachment of vesicles to selected cells, the cells were first labeled with biotin-modified antibody then treated with sealed ghosts prepared from avidin-coupled erythrocytes. This procedure should prove useful when the injection of specific cell populations is desired, or with cell types such as lymphocytes that are difficult to fuse, or when the use of limited reagents necessitates high injection efficiencies.Microinjection allows the introduction into cells of biologically interesting molecules so that their properties and effects on cell function can be studied. Although microinjection protocols have been used to study a number of important questions in cell biology (1-4), their applications have been restricted by inherent limitations. Mechanical injection (5) can be used to introduce discrete volumes of reagent into cells, but it is limited by the number and type of cells that can be treated (1). Vesicle-mediated protocols, using fusogens such as polyethylene glycol (PEG) or Sendai virus, can be used to inject large numbers of cells (1), but fusion efficiencies are generally low and selectivity is lacking.We, as others (6), have reasoned that bringing two membranes into close apposition should increase the probability that they will fuse, although contact per se between two membranes does not generally lead to fusion (7). PEG-mediated microinjection can be enhanced by agglutinating vesicles to cells with phytohemagglutinin (6, 8). Our laboratory has developed protocols for the efficient, antibody-directed targeting of vesicles to cell surface antigens (9). In this paper, we describe the use of vesicle targeting with PEG-mediated fusion to promote the microinjection of selected cell types, particularly of lymphocytes and lymphoid cell lines. The frequency of vesicle-lymphocyte fusions in these experiments is about 104 times higher than values generally observed for PEG-mediated lymphocyte fusion. MATERIALS AND METHODSAntibodies. Goat anti-mouse immunoglobulin (anti-MIg) was raised as described (10). Anti-mouse cell surface (anti-MCS) was prepared by injecting rabbits with DBA/2 spleen and lymph node cells in complete Freund's adjuvant. Rabbit anti-mouse Fab fragment (anti-MFab) was generously donated by A. Good (University of California, Berkeley). All three antisera were passed over DEAE-Sephacel (Pharmacia). Fab fragment of antiMIg (11) and the affinity-purified F(ab')2 fragment of anti-MFab (9) were prepared as described. Two monoclonal antibodies were obtained from Becton Dickinson: mouse ant...

Flow cytometry and fluorescence microscopy both provide single-cell analysis using different but complementary sets of data, essentially population-based target intensities versus target morphology in relatively small sample sizes. Both approaches generally employ optical filters to analyze fluorescence emissions, and have to overcome some of the same physical limitations, including spectral overlap of dyes and the dynamic range limits of measuring systems. Some of the technical challenges differ: dye photostability is more critical to microscopy; creating suspensions from adherent cells can impact flow cytometric analysis. With extensive image acquisition and processing, the microscopist may arrive at quantitative data. However, the cooperative use both flow cytometry and microscopy can provide more robust interrogation of biological phenomena.A number of examples will be presented to illustrate both the complementarity that microscopy and flow cytometry perspectives bring to new applications and ultimately, biological questions. They also illustrate the constraints to migrating assays between platforms. Viability assays based on membrane integrity or esterase activity generally provide bright, well-resolved signals easily read by microscopy or flow cytometry (Figures 1-2). Microscopy has helped verify mitochondrial specificity and responsiveness with mitochondrial function assays that can be quantified by flow cytometry. However, assays that depend on translocation rather than overall intensity change, such as loss of cytochrome c from mitochondria, are better performed by microscopy. Flow cytometry can readily quantify live cells in various stages of cell cycle using a number of cell-permeant DNA dyes; microscopy confirms nuclear labeling and also indicates that some of these dyes may have adverse morphological affects. Kinetic studies, such as calcium response, can be accomplished on individual cells by microscopy, and easily quantified on populations by flow cytometry. Morphological information, as with organelle structure, is readily available through microscopy, but flow cytometry may provide little useful information about morphology. One example of complementarity between microscopy and flow cytometry is provided by analysis of mitochondrial DNA (mtDNA) depletion. Microscopy has demonstrated the decrease of mtDNA-encoded COXI, including mosaicism, in individual fibroblasts following progressive exposure to anti-retroviral drugs. Flow cytometry has provided rapid population statistics to support the visual results. 1,2Fluorescence microscopy is well suited to the resolution of cell and tissue architecture, and to following kinetic and trophic responses in single cells. Flow cytometry rapidly quantifies small differences between cell populations using statistically-significant numbers of events. Flow cytometry can represent a "black box" when looking at the magnitude of a population response; fluorescence microscopy can help verify that measured results represent meaningful biological effects.