High-speed Cell Sorting Research Articles

Early-Senescing Human Skin Fibroblasts Do Not Demonstrate Accelerated Telomere Shortening

Most normal mammalian cell lines demonstrate limited growth capacity due to the gradual accumulation of senescent cells in the culture. Senescent cells appear initially at a low incidence, but with increasing frequency as the culture accumulates more divisions. Because it has been suggested that senescence is regulated by telomere shortening in human cells, we compared the telomere lengths of the subpopulation of senescent cells, present in presenescent cultures, with those of young cells. Senescent cells were separated from young cycling cells by either bromodeoxyuridine (BrdU) incorporation followed by Hoechst dye and light treatment or DiI staining followed by separation on a high-speed cell sorter. Our results demonstrate that telomeres of early-senescing cells are the same length, and must shorten at the same rate, as cycling sister cells in the culture. Therefore, senescent cells in young mass cultures occur as a result of a stochastic, nontelomere-dependent process that we have described: sudden senescence syndrome.

A dual-fluorescence reporter system for high-throughput clone characterization and selection by cell sorting.

Molecular biology critically depends upon the isolation of desired DNA sequences. Flow cytometry, with its capacity to interrogate and sort more than 50 000 cells/s, shows great potential to expedite clone characterization and isolation. Intrinsic heterogeneity of protein expression levels in cells limits the utility of single fluorescent reporters for cell-sorting. Here, we report a novel dual-fluorescence strategy that overcomes the inherent limitations of single reporter systems by controlling for expression variability. We demonstrate a dual-reporter system using the green fluorescent protein (GFP) gene fused to the Discosoma red fluorescent protein (DsRed) gene. The system reports the successful insertion of foreign DNA with the loss of DsRed fluorescence and the maintenance of GFP fluorescence. Single cells containing inserts are readily recognized by their altered ratios of green to red fluorescence and separated using a high-speed cell-sorter for further processing. This novel reporter system and vector were successfully validated by shotgun library construction, cloned sequence isolation, PCR amplification and DNA sequencing of cloned inserts from bacteria after cell-sorting. This simple, robust system can also be adapted for diverse biosensor assays and is amenable to miniaturization. We demonstrated that dual-fluorescence reporting coupled with high-speed cell-sorting provides a more efficient alternative to traditional methods of clone isolation.

Production of Lambs After the Transfer of Fresh and Cryopreserved in vitro Produced Embryos from Prepubertal Lamb Oocytes and Unsorted and Sex‐sorted Frozen‐thawed Spermatozoa

Cumulus-oocyte complexes from hormone-stimulated 3-4-week-old (n=43) and 6-7-week-old (n=12) prepubertal lambs were matured in vitro and incubated with unsorted, or X- or Y-spermatozoa separated with a high-speed cell sorter (SX MoFlo)frozen-thawed. Presumptive zygotes were then cultured to the blastocyst stage, and transferred to recipients fresh or after cryopreservation (frozen). Oocyte cleavage was higher (p <0.05) with unsorted (515/926, 55.6%) than X- or Y-spermatozoa (261/672, 38.8% and 229/651, 35.2%, respectively) and blastocyst formation (% zygotes) by Day 9 of in vitro culture was lower (p <0.05) for X- (102/261, 39.1%) than unsorted spermatozoa (249/515, 48.3%), but did not differ between Y-spermatozoa (103/229, 45.0%) and unsorted spermatozoa, or between X- and Y-spermatozoa (p >0.05). For fresh embryos, survival to term was 50.0% (3/6) for unsorted, 0.0% (0/6) for X- and 16.7% (1/6) for Y-spermatozoa-derived embryos (p >0.05), and for frozen embryos was 4.0% (2/50) for unsorted, 9.1% (2/22) for X- and 2.9% (1/34) Y-spermatozoa-derived embryos (p >0.05). Of the two lambs born from X-spermatozoa-derived embryos, one was female (50%), and from the two Y-spermatozoa-derived lambs, both were male (100%), demonstrating that lambs can be produced after the transfer of fresh and cryopreserved IVP embryos derived from prepubertal lamb oocytes and frozen-thawed sex-sorted sperm.

Isolation and tracking of a rare lymphoid progenitor cell which facilitates bone marrow transplantation in mice

Bone marrow cells are composed of pluripotent stem cells to terminally differentiated cells, with a wide variety of abundance of each cell type. In the past, many of the cell types within this heterogeneous population have been characterized either by expression of specific proteins or using functional markers. In spite of promising results obtained with the latter method, various cell types within bone marrow have not been well characterized due to the low abundance of a specific cell type. Considering the demand for a reliable technique to enrich cell types, a wide variety of approaches, ranging from simple nylon wool columns to high-speed cell sorting, have evolved. Only limited success has been obtained with approaches ranging from the detection of MHC antigen to positron emission tomography to track the ontogeny of specific bone marrow-derived cells in studies of syngeneic or allogeneic transplantation. The present study describes a relatively simple method to enrich and track a rare bone marrow cell (facilitating cell, FC), which can facilitate allogeneic bone marrow stem cell transplantation in mice. The isolation technique is comprised of enrichment of FC by magnetic activated cell sorting (MACS) system followed by purification through high-speed cell sorter. An initial inoculation of 30,000 FC obtained from male mice was detected in the thymus, spleen, and bone marrow of allogeneic female recipients, by using 32P-labeled dCTP in a specific PCR for Y-chromosome. This technique may improve the efficiency of isolation of other rare cells from the bone marrow.

Flow cytometric sorting of non-human primate sperm nuclei

Pre-determination of the sex of offspring has implications for management and conservation of captive wildlife species, particularly those with single sex-dominated social structures. Our goal is to adapt flow cytometry technology to sort spermatozoa of non-human primate species for use with assisted reproductive technologies. The objectives of this study were to: (i) determine the difference in DNA content between X- and Y-bearing spermatozoa (ii) sort sperm nuclei into X- and Y-enriched samples; and (iii) assess the accuracy of sorting. Spermatozoa were collected from two common marmosets ( Callithrix jacchus), seven hamadryas baboons ( Papio hamadryas) and two common chimpanzees ( Pan troglodytes). Human spermatozoa from one male were used as a control. Sperm nuclei were stained (Hoechst 33342), incubated and analyzed using a high-speed cell sorter. Flow cytometric reanalysis of sorted samples (sort reanalysis, 10,000 events/sample) and fluorescence in situ hybridization (FISH; 500 sperm nuclei/sample) were used to evaluate accuracy of sorting. Based on fluorescence intensity of X- and Y-bearing sperm nuclei, the difference in DNA content between X and Y populations was 4.09 ± 0.03, 4.20 ± 0.03, 3.30 ± 0.01, and 2.97 ± 0.05%, for marmoset, baboon, chimpanzee and human, respectively. Sort reanalysis and FISH results were similar; combined data revealed high levels of purity for X- and Y-enriched samples (94 ± 0.9 and 93 ± 0.8%, 94 ± 0.7 and 94 ± 0.5%, 91 ± 0.9 and 97 ± 0.6%, 94 ± 0.6 and 94 ± 0.9%, for marmoset, baboon, chimpanzee and human, respectively). These data indicate the potential for high-purity sorting of spermatozoa from non-human primates.

Assessment of in vitro sperm characteristics after flow cytometric sorting of frozen-thawed bull spermatozoa

The effect of processing prior to sex-sorting, re-freezing and thawing of frozen-thawed bull spermatozoa on in vitro sperm characteristics was investigated. Frozen-thawed bull spermatozoa (three bulls; three ejaculates per bull) were prepared for sorting by washing (FT-WASH) or gradient centrifugation (FT-GRADIENT) and evaluated for motility and forward progressive motility (FPM) after processing, staining, sorting and incubation (3 h; 37 °C). After frozen-thawed samples were processed and analyzed using a high-speed cell sorter, aliquots were removed and re-frozen and thawed (FTF-WASH; FTF-GRADIENT). Non-sorted frozen-thawed spermatozoa (FT-CONTROL) were also re-frozen and thawed (FTF-CONTROL). Spermatozoa from all treatments were assessed for penetration of an artificial cervical mucus at 0 h after sorting or thawing, and for motility, FPM and acrosomal status after 3-h incubation (37 °C). Frozen-thawed spermatozoa prepared by gradient centrifugation before sorting were sorted more efficiently than washed samples ( P<0.05). However, after sorting (FT) or thawing (FTF) and incubation, the percentage of motile spermatozoa and FPM rating was lower for GRADIENT than WASH (21.5±3.39%; 1.4±0.16 FPM versus 48.6±4.02%, 2.6±0.16 FPM; P<0.01). Frozen-thawed sorted spermatozoa (FT) penetrated in greater numbers (151.0±19.50 spermatozoa) and distance (56.3±5.11 mm) in the artificial cervical mucus and had a higher proportion of motile spermatozoa (65.5±2.77%) and FPM rating (2.8±0.12) after incubation than spermatozoa that had been re-frozen and thawed after sorting (FTF: 14.0±3.67 spermatozoa, 21.6±3.05 mm, 12.2±1.31% and 1.2±0.10 FPM, respectively; P<0.001). Regardless of processing prior to sorting, frozen-thawed sorted and non-sorted spermatozoa migrated similar distances in the artificial cervical mucus (FT-WASH: 60.0±1.2 mm; FT-GRADIENT: 57.2±0.76 mm; FT-CONTROL: 51.7±0.69 mm). The results of this preliminary study suggested that frozen-thawed bull spermatozoa can be efficiently sorted into high purity X- and Y-chromosome enriched samples with retained functional capacity.

Optical monitor for measuring the amplitude and phase of perturbations on the surface of a capillary jet in a high-speed cell sorter

High-speed jet-in-air cell sorters analyze and sort thousands of cells a second. The key to the accuracy and purity of their operation is the ability to maintain a stable location on the jet where the stream breaks into individual droplets. The physics of the instability of a capillary jet are well understood and date back to the 19th century. The time it takes for a jet of liquid to break into droplets depends exponentially on the amplitude of perturbations that develop on the surface of the jet. In this work, we describe a method to monitor the amplitude of these perturbations on the surface of a liquid jet using a novel detector. This detector may be combined with feedback mechanisms to actively stabilize the break off point in modern cell sorters. We also show that this detector can accurately monitor the phase of the perturbations with respect to the driving element, and this phase information, in turn, can be used to improve sort rates for high-throughput cell sorters.

High-speed chromosome sorting.

Structural and genetic characterization of chromosomes is necessary to understand both normal and pathologic physiology in any species. Flow cytometry and cell sorting technologies provide a means for precise measurement of chromosomal makeup as well as for the isolation of specific chromosomes for further study. Advancements in molecular biology protocols and pressures from large-scale sequencing endeavors placed increased demand on the developers of these instruments for enhanced throughput and quality of results. The ensuing improvements in sorting performance led to the development of a new generation of cytometers known as high-speed cell sorters. These machines provide superior results in less time and are cheaper and simpler to operate than their predecessors. Robust chromosome sorting can now be performed in the laboratories of individual investigators for a variety of gene- and sequence-specific studies. Resolution of the flow karyotype with increased refinement, and the development of new applications for this technology will assure that cell sorting continues to play an important role in cytogenetics, our understanding of molecular processes such as evolution and disease etiology, and ultimately serve as a launching point for predictive medicine.

Viable infectious cell sorting in a BSL-3 facility.

With the increase in demand for high-speed cell sorting of viable infectious and now therapeutic cell samples, safety concerns for the protection of flow cytometer operators have increased. This chapter describes a quick, sensitive, and reproducible procedure to assure sample containment before sorting these samples. This procedure includes aerosol containment, physical barriers, environmental controls, and personal protection. An aerosol management system produces a negative pressure within the sort chamber where aerosols are vacuumed directly into a HEPA filter. Physical barriers include the manufacturer's standard plastic shield and panels. The flow cytometer is contained in a BSL-3 laboratory for maximum environmental control and the operator is protected using a respiratory system. Containment is measured using highly fluorescent Glo-Germ particles under the same conditions as the cell sort but with the sorter adjusted to produce large amounts of aerosols. These aerosols are collected by a vacuum air sampling system for 10 min in three locations onto a glass slide and examined microscopically. With this system in place, aerosol containment can be measured quickly and efficiently, therefore reducing the risk to the operator when sorting viable infectious cells.

Stability of the breakoff point in a high-speed cell sorter.

High-speed jet-in-air cytometric sorting requires knowledge of the time it takes a particle to travel from the laser to the point where the jet breaks into droplets. Variations in this breakoff time will result in poorer yields and poorer sort purities. This work examined the physical mechanisms that lead to the break up of the jet into droplets and calculated the stability of the droplet breakoff time relative to physical parameters, which govern the behavior of the jet. We derived the variations in the breakoff time and found that small variations in the drive frequency, temperature, pressure, and drive amplitude can lead to correspondingly large changes in the breakoff time. We found explicitly that the time it takes the jet to break up is not necessarily correlated with the distance to the breakoff point. Many high-speed cell sorters use active means to control the breakoff time. A common method to monitor the breakoff time is to visually monitor the breakoff point. This technique in fact may decrease the sorting purity and efficiency by inadvertently correcting for breakoff time variations. We show explicitly the breakoff time's dependence on a number of physical parameters that can be monitored to increase the stability of the breakoff time.

Rapid expression cloning of receptors using epitope-tagged ligands and high-speed cell sorting.

In this study we describe a new approach for expression cloning of receptors. Our approach was based on highly efficient transfer of retroviral cDNA libraries into target cells and detection of receptor-ligand interaction with the use of an antibody directed against an epitope tag on recombinant ligands. Detection of the complex and isolation of receptor-transduced cells were achieved by flow cytometry and rare event high-speed cell sorting. Recovery of the cDNA coding for the receptor(s) was achieved by polymerase chain reaction. As a proof-of-concept study we set out to clone the receptor for B-lymphocyte stimulator protein (BlyS), not known at the start of the project but reported while this work was in progress. First, we detected binding of epitope-tagged BlyS to IM9 cells. Second, human T-lymphoblasts (CEM cells), which do not bind BlyS, were transduced with a retroviral cDNA library generated from IM9 cells. Transduced CEM cells binding epitope-tagged BlyS protein were identified by flow cytometry. After three sequential rounds of cell sorting, transduced CEM cell populations with high binding capacity for BlyS were identified. To determine the cDNAs conferring binding to the transduced CEM cells, the integrated proviral DNAs were amplified by polymerase chain reaction and analyzed by DNA sequencing. Rescued cDNAs contained Transmembrane Activator and calcium-modulator and cyclophilin ligand (CAML) Interactor (TACI) and B-Cell Maturation factor (BCMA) sequences, representing two published receptors of BlyS. Our data demonstrated that flow cytometry and high-speed cell sorting combined with transduction of retroviral cDNA libraries and binding of epitope-tagged orphan ligands as a selectable phenotype can be used efficiently for expression cloning of receptors. Of particular interest was our finding that apparently it is not necessary to purify the ligand but that conditioned medium containing the ligand can be used instead. Thus we concluded that our approach shortens the time to identify receptors for many orphan ligands and helps to exploit these receptors as drug targets.

Flow cytometric sorting of frozen-thawed spermatozoa in sheep and non-human primates.

Research was conducted in sheep to determine an effective preparation method for high-purity sorting of frozen-thawed spermatozoa. The efficacy of sorting frozen-thawed spermatozoa was then investigated in several non-human primate species. An aliquot of each ejaculate (three rams, three ejaculates per ram) was processed as a fresh control (FRESH). Frozen spermatozoa were thawed and prepared for sorting by no further processing (FT-NEAT), washing (FT-WASH) or gradient centrifugation (FT-GRADIENT) and evaluated for motility at 1 h post-staining and motility and acrosomal status at 0 and 4 h post-sorting. Samples were analysed using a high-speed cell sorter. High levels of purity for X- and Y-enriched samples were achieved for all treatments (85-92%). The percentage of motile spermatozoa before sorting was lower (P < 0.05) for frozen-thawed samples (FT-NEAT: 32.7 +/- 2.5%; FT-WASH: 32.2 +/- 3.3%; FT-GRADIENT: 73.9 +/- 3.7%) compared with FRESH (83.3 +/- 1.2%). Post-sorting, the percentage of motile spermatozoa before and after incubation for FT-NEAT (60.0 +/- 5.1% and 27.2 +/- 6.1% for 0 and 4 h, respectively) was lower than that for FRESH (87.8 +/- 0.9% and 83.3 +/- 1.2% for 0 and 4 h, respectively; P < 0.05), FT-WASH (80.0 +/- 2.4% and 71.7 +/- 3.6% for 0 and 4 h, respectively; P < 0.05) and FT-GRADIENT (84.4 +/- 1.3% and 77.2 +/- 1.7% for 0 and 4 h, respectively; P < 0.05). Vanguard sperm migration distance through artificial cervical mucus was lower (P < 0.05) for FT-NEAT (17.7 +/- 1.7 mm) compared with FT-WASH (29.1 +/- 3.8 mm) and FT-GRADIENT (28.4 +/- 2.0 mm) and similar (P < 0.05) to FRESH (23.7 +/- 1.8 mm). Sample preparation using a modified wash method enabled high-purity sorting (range 86-97% purity) of frozen-thawed epididymal spermatozoa in the baboon (Papio hamadryas), common marmoset (Callithrix jacchus) and common chimpanzee (Pan troglodytes). For all non-human primate species, sorted spermatozoa were progressively motile (marmoset: 20.5 +/- 5.5%; baboon: 37.5 +/- 2.5%; chimpanzee: 73.0 +/- 2.0%), acrosome intact (marmoset: 68.5 +/- 7.5%; baboon: 89.5 +/- 1.5%; chimpanzee: 84.0 +/- 1.0%) and able to penetrate an artificial cervical mucus. In summary, high-purity sorting of frozen-thawed ram and non-human primate spermatozoa with recovery of progressively motile, acrosome-intact spermatozoa was possible after processing to remove cryodiluent.

Homing of purified murine lymphohematopoietic stem cells: a cytokine-induced defect.

This study was designed to establish a direct homing assay using purified lineage-negative Sca-1-positive (Lin(-) Sca(+)) murine bone marrow cells and to evaluate the effects of cytokines on homing. C57BL/6 Lin(-) Sca(+) marrow stem cells were labeled with 5-(and 6)-carboxyfluorescein diacetate succinimidyl ester (CFDA-SE) and then injected by tail vein into untreated C57BL/6 mice. Marrow was harvested at various times after cell infusion and analyzed on a high-speed MoFlo cell sorter for fluorescent positive events, using a large event analysis, with at least 16 million total events analyzed. We have shown that homing of Lin(-) Sca(+) cells plateaus by 1 h, and at 3 h post-infusion is linear between 50,000 and 1,000,000 infused cells. This forms a base for a homing assay in which 250,000 CFDA-SE labeled Lin(-) Sca(+) marrow cells are infused and then recovered from marrow 3 h later, followed by a large-event fluorescence-activated cell sorting (FACS) analysis. We found that 7.45-9.32% of infused cells homed and that homing of stem cells cultured for 48 h in interleukin-3 (IL-3), IL-6, IL-11, and steel factor cultured cells was defective when compared to noncultured cells. Exposure of marrow stem cells to IL-3, IL-6, IL-11, and steel factor induces a stem cell homing defect, which probably underlies the engraftment defect previously characterized under these conditions.

Basic aspects of high-speed sorting for clinical applications

High-speed cell sorting is currently being used in a number of clinical applications due to its obvious benefits of speed and purity. These applications include rare-cell sorting for the enrichment of stem cells, DCs, fetal cells in maternal circulation, Ag-specific T cells, as well as tumor-cell detection and enrichment for residual disease diagnosis, or depletion for transplant purging. Highspeed sorting is not limited to rare populations, as sperm sorting for gender selection demonstrates. Operators performing clinical flow sorting may come into contact with potentially infectious material. Since cell sorters can generate aerosols, aerosol-containment systems should be used for instrument-operator protection. In addition, if flow sorting is to be used to purify cell populations for reinfusion into patients, a more rigorous set of precautions is needed to protect the sample during this process. Cell sorter manufacturers have devised equipment to help with these goals, and the solutions currently available for the Cytomation (Fort Collins, Colorado) MoFlo high-speed cell sorter were presented. Overview of high-speed sorting technology There are requirements of both the instrumentation and cellular sample for high-speed cell sorting. Instrumentation should be designed at the outset with this capacity in mind. Cellular samples must be at a high concentration in order for the machine to properly utilize such a system, allowing a high-speed train of single cells to be presented. Instrumentation can also be used for conventional sorting (, 10 000 event/s), with cell samples at lower concentrations giving excellent results.

Isolation of Hair Mesenchymal Cells by High-Speed Cell Sorting and Verification of Their Inductive Ability on Hair Follicle Reconstitution.

Isolation of Hair Mesenchymal Cells by High-Speed Cell Sorting and Verification of Their Inductive Ability on Hair Follicle Reconstitution.

Production of lambs of predetermined sex after the insemination of ewes with low numbers of frozen-thawed sorted X- or Y-chromosome-bearing spermatozoa.

The fertilizing ability of sex-sorted frozen-thawed ram spermatozoa was assessed after insemination of mature Merino ewes at a synchronized oestrus. Ewes were inseminated into the uterus or utero-tubal junction (UTJ) with a total of 140 x 10(6) unsorted (control) or 2-4 x 10(6) sorted (X or Y) frozen-thawed ram spermatozoa 54 to 57 hours after removal of progestagen-impregnated pessaries and an injection of 400 IU of pregnant mare serum gonadotrophin (Folligon, Intervet). The spermatozoa were separated into X- and Y-chromosome-bearing spermatozoa after analysis with a modified high-speed cell sorter (SX MoFlo). The number of ewes pregnant after insemination with unsorted frozen-thawed spermatozoa was significantly higher (26/48; 54.3%) than for ewes inseminated with either X- (12/48; 25.0%) or Y-sorted spermatozoa (7/48; 14.6%) (P<0.05). Seventeen of the eighteen lambs produced by ewes inseminated with X-sorted spermatozoa were female (94.4%) and 8/8 lambs from ewes inseminated with Y-sorted spermatozoa were male (100%). The sex ratio of the lambs born to ewes inseminated with sex-sorted spermatozoa was significantly skewed from the 51.3% male and 48.7% female ratio in the control group (P<0.05). This study showed, for the first time, that lambs of predicted sex can be produced after insemination with low numbers of sex-sorted cryopreserved ram spermatozoa.

Genomics and proteomics in cancer: Proceedings of the 3rd Samuel A. Latt conference, Detroit, Michigan

Genomics and proteomics in cancer: Proceedings of the 3^rd Samuel A. Latt conference, Detroit, Michigan

Transplantation with selected autologous peripheral blood CD34 +Thy1 + hematopoietic stem cells (HSCs) in multiple myeloma : Impact of HSC dose on engraftment, safety, and immune reconstitution

Objective The aims of our study performed in myeloma were to evaluate the performance and the safety of Systemix's high-speed clinical cell sorter, to assess the safety and efficacy of de-escalating cell dose cohorts of CD34 +Thy1 + hematopoietic stem cells (HSCs) as autologous grafts by determining engraftment, and to assess the residual tumor cell contamination using polymerase chain reaction (PCR) amplification assays of patient-specific complementarity determining region III (CDR III) analysis for residual myeloma cells. Materials and Methods The clinical trial was performed in 31 multiple myeloma patients, using purified human CD34 +Thy1 + HSCs mobilized from peripheral blood with cyclosphosphamide and granulocyte-macrophage colony-stimulating factor to support a single transplant after high-dose melphalan 140 mg/m 2 alone (cohort 1) and with total body irradiation (TBI) (cohorts 2–5) after an HSC transplant cell dose de-escalation/escalation design. Results Twenty-three patients were transplanted. Engraftment data in the melphalan + TBI cohorts confirmed that HSC doses above the threshold dose of 0.8 × 10 6 CD34 +Thy1 + HSCs/kg provided prompt engraftment (absolute neutrophil count >0.5 × 10 9/L day 10; platelet count >50 × 10 9/L day 13). A higher rate of infections was observed in the early and late follow-up phases than usually reported after CD34 + selected or unselected autologous transplantation, which did not correlate with the CD34 +Thy1 + HSC dose infused. Successful PCR for CDR III could only be performed in five patients on initial apheresis product and final CD34 +Thy1 + HSC product and showed a median tumor log reduction >3.12. Conclusions CD34 +Thy1 + HSCs are markedly depleted or free of detectable tumor cells in multiple myeloma and are capable of producing fast and durable hematopoietic reconstitution at cell doses >0.8 × 10 6 CD34 +Thy1 + HSCs/kg. The delayed immune reconstitution observed is not different from that described in unselected autologous bone marrow and peripheral blood mononucleated cells transplants in multiple myeloma and may be corrected by addition of T cells either to the graft or to the patient in the posttransplant phase.

Col2-GFP reporter marks chondrocyte lineage and chondrogenesis during mouse skeletal development.

Mice were generated in which a Col2-GFP transgene serves as a reporter for the chondrocyte lineage and for chondrogenesis in live embryos and newborn pups. Cells actively engaged in chondrogenesis were identified by confocal optical sectioning within their native environments in embryos and in thick tissue slices. Chondrocytes exhibiting GFP fluorescence were purified from rib cages by high-speed cell sorting of crude cell suspensions. Intensity of fluorescence correlated with biosynthesis of procollagen II in these cells. The use of these mice and their cells provides a novel approach for studying chondrocyte differentiation and chondrogenesis during skeletal development.

Sex preselection: High-speed flow cytometric sorting of X and Y sperm for maximum efficiency

Sex preselection that is based on flow-cytometric measurement of sperm DNA content to enable sorting of X- from Y-chromosome-bearing sperm has proven reproducible at various locations and with many species at greater than 90% purity. Offspring of the predetermined sex in both domestic animals and human beings have been born using this technology since its introduction in 1989. The method involves treating sperm with the fluorescent dye, Hoechst 33342, which binds to the DNA and then sorting them into X- and Y-bearing-sperm populations with a flow cytometer/cell sorter modified specifically for sperm. Sexed sperm are then used with differing semen delivery routes such as intra-uterine, intra-tubal, artificial insemination (deep-uterine and cervical), in vitro fertilization and embryo transfer, and intra-cytoplasmic sperm injection (ICSI). Offspring produced at all locations using the technology have been morphologically normal and reproductively capable in succeeding generations. With the advent of high-speed cell sorting technology and improved efficiency of sorting by a new sperm orienting nozzle, the efficiency of sexed sperm production is significantly enhanced. This paper describes development of the these technological improvements in the Beltsville Sexing Technology that has brought sexed sperm to a new level of application. Under typical conditions the high-speed sperm sorter with the orienting nozzle (HiSON) results in purities of 90% of X- and Y-bearing sperm at 6 million sperm per h for each population. Taken to its highest performance level, the HiSON has produced X-bearing-sperm populations at 85 to 90% purity in the production of up to 11 million X- bearing-sperm per h of sorting. In addition if one accepts a lower purity (75 to 80%) of X, nearly 20 million sperm can be sorted per h. The latter represents a 30 to 60-fold improvement over the 1989 sorting technology using rabbit sperm. It is anticipated that with instrument refinements the production capacity can be improved even further. The application of the current technology has led to much wider potential for practical usage through conventional and deep-uterine artificial insemination of many species, especially cattle. It also opens the possibility of utilizing sexed sperm for artificial insemination in swine once low-sperm-dose methods are perfected. Sexed sperm on demand has become a reality through the development of the HiSON system.