High-speed Cell Sorting Research Articles

Rapid Isolation of Antibody from a Synthetic Human Antibody Library by Repeated Fluorescence-Activated Cell Sorting (FACS)

Antibodies and their derivatives are the most important agents in therapeutics and diagnostics. Even after the significant progress in the technology for antibody screening from huge libraries, it takes a long time to isolate an antibody, which prevents a prompt action against the spread of a disease. Here, we report a new strategy for isolating desired antibodies from a combinatorial library in one day by repeated fluorescence-activated cell sorting (FACS). First, we constructed a library of synthetic human antibody in which single-chain variable fragment (scFv) was expressed in the periplasm of Escherichia coli. After labeling the cells with fluorescent antigen probes, the highly fluorescent cells were sorted by using a high-speed cell sorter, and these cells were reused without regeneration in the next round of sorting. After repeating this sorting, the positive clones were completely enriched in several hours. Thus, we screened the library against three viral antigens, including the H1N1 influenza virus, Hepatitis B virus, and Foot-and-mouth disease virus. Finally, the potential antibody candidates, which show KD values between 10 and 100 nM against the target antigens, could be successfully isolated even though the library was relatively small (∼106). These results show that repeated FACS screening without regeneration of the sorted cells can be a powerful method when a rapid response to a spreading disease is required.

Two functionally distinct myeloid dendritic cell subpopulations are present in bovine blood

Immature myeloid (m)DCs circulating in the blood of cattle have been defined as lineage negative (Lin−)MHCII+CD11c+CD205+ cells. Lin−MHCII+CD11c+CD205+ mDCs (0.2% blood mononuclear cells) isolated from bovine blood were heterogeneous in cell size and CD205 expression. Using highspeed cell sorting, Lin−MHCII+CD11c+CD205+ DCs were sorted into CD205Hi and CD205Lo subpopulations which were phenotypically distinct and differed significantly (P<0.01) in TLR gene expression. CD205Hi and CD205Lo mDCs were more efficient in macropinocytosis than monocytes and expressed no or little detectable non-specific esterase activity. CD205Lo mDCs efficiently activated purified allogeneic T cells and the addition of TLR agonists did not significantly alter this antigen presentation capacity. T cell activation by CD205Lo mDCs was associated with differential up-regulation of CD40, CD80, CD86 and TGFβ1 gene expression when compared to CD205Hi mDCs. In conclusion, two phenotypically and functionally distinct CD11c+CD205+ mDCs were isolated from blood that had an equal capacity to acquire antigen but markedly different capacities to activate T cells.

Mitochondrial Staining Allows Robust Elimination of Apoptotic and Damaged Cells during Cell Sorting

High-speed fluorescence-activated cell sorting is relevant for a plethora of applications, such as PCR-based techniques, microarrays, cloning, and propagation of selected cell populations. We suggest a simple cell-sorting technique to eliminate early and late apoptotic and necrotic cells, with good signal-to-noise ratio and a high-purity yield. The mitochondrial potential dye, TMRE (tetramethylrhodamine ethyl ester perchlorate), was used to separate viable and non-apoptotic cells from the cell sorting samples. TMRE staining is reversible and does not affect cell proliferation and viability. Sorted TMRE(+) cells contained a negligible percentage of apoptotic and damaged cells and had a higher proliferative potential as compared with their counterpart cells, sorted on the basis of staining with DNA viability dye. This novel sorting technique using TMRE does not interfere with subsequent functional assays and is a method of choice for the enrichment of functionally active, unbiased cell populations.

Growth and Airborne Transmission of Cell-Sorted Life Cycle Stages of Pneumocystis carinii

Pneumocystis organisms are airborne opportunistic pathogens that cannot be continuously grown in culture. Consequently, the follow-up of Pneumocystis stage-to-stage differentiation, the sequence of their multiplication processes as well as formal identification of the transmitted form have remained elusive. The successful high-speed cell sorting of trophic and cystic forms is paving the way for the elucidation of the complex Pneumocystis life cycle. The growth of each sorted Pneumocystis stage population was followed up independently both in nude rats and in vitro. In addition, by setting up a novel nude rat model, we attempted to delineate which cystic and/or trophic forms can be naturally aerially transmitted from host to host. The results showed that in axenic culture, cystic forms can differentiate into trophic forms, whereas trophic forms are unable to evolve into cystic forms. In contrast, nude rats inoculated with pure trophic forms are able to produce cystic forms and vice versa. Transmission experiments indicated that 12 h of contact between seeder and recipient nude rats was sufficient for cystic forms to be aerially transmitted. In conclusion, trophic- to cystic-form transition is a key step in the proliferation of Pneumocystis microfungi because the cystic forms (but not the trophic forms) can be transmitted by aerial route from host to host.

Abstract 566: Angiotensin II Promotes Proliferation and Fibrosis in Parietal Epithelial Cells Contributing to the Development of Crescentic Glomerulonephritis

Introduction: Parietal epithelial cells (PECs) contribute to the glomerular crescent formation of crescentic glomerulonephritis. Co-cultured model consisting of mesangial cells (MCs), PECs, and macrophages which play a pivotal role in the development of crescentic glomerulonephritis showed an increase in Angiotensin (Ang) II-induced cell proliferation and collagen secretion in PECs. Moreover, Ang II type 1 receptor blocker (ARB) treatment completely prevented an increase in cell proliferation and collagen secretion in PECs under the co-cultured model. However, it is not clear whether Ang II directly promotes PEC fibrosis and proliferation under the monoculture in PECs. Aim: The aim of present study is to demonstrate that Ang II stimulation directly affects PEC proliferation and fibrosis. Methods: Primary fluorescein isothiocyanate (FITC)-labeled PEC population was collected from glomeruli in rats using high-speed cell sorter. ERK1/2 phosphorylation was evaluated by Western blotting. Cell proliferation ability was measured by water-soluble tetrazolium salts (WST)-1 and the soluble collagen levels in culture supernatants was determined by the Sircol collagen assay. Results: FITC-labeled PECs could be identified as a distinct cell population by high-speed cell sorter. The ERK 1/2 phosphorylation in PECs was stimulated by Ang II (100 nmol/l) by about 1.7-fold. After the treatment with 100 nmol/l Ang II for 24 hrs, cell proliferation ability significantly increased in PECs (mean ± SEM: 1.60 ± 0.03 vs. 1.00 ± 0.04, relative ratio). However, the proliferation ability in Ang II-stimulated PECs was suppressed by Ang II plus 10 nmol/l olmesartan (an ARB) or Ang II plus 100 nmol/l PD98059 (a MEK inhibitor). PECs also promoted a collagen secretion after the stimulation by 100 nmol/l Ang II for 24 hrs (52.1 ± 8.1 vs. 16.8 ± 2.4 μg/ml). Furthermore, the significant decrease in soluble collagen secretion was observed by the treatments with Ang II plus 10 nmol/l olmesartan or Ang II plus 10 μg/ml pan-specific neutralizing TGF-β antibody. Conclusion: These data indicate that Ang II-stimulated PECs promote proliferation and fibrosis and may suggest contributing to the development of crescentic glomerulonephritis by PECs independently from MCs or macrophages.

A Transcriptomic Network Identified in Uninfected Macrophages Responding to Inflammation Controls Intracellular Pathogen Survival

SummaryIntracellular pathogens modulate host cell function to promote their survival. However, in vitro infection studies do not account for the impact of host-derived inflammatory signals. Examining the response of liver-resident macrophages (Kupffer cells) in mice infected with the parasite Leishmania donovani, we identified a transcriptomic network operating in uninfected Kupffer cells exposed to inflammation but absent from Kupffer cells from the same animal that contained intracellular Leishmania. To test the hypothesis that regulated expression of genes within this transcriptomic network might impact parasite survival, we pharmacologically perturbed the activity of retinoid X receptor alpha (RXRα), a key hub within this network, and showed that this intervention enhanced the innate resistance of Kupffer cells to Leishmania infection. Our results illustrate a broadly applicable strategy for understanding the host response to infection in vivo and identify Rxra as the hub of a gene network controlling antileishmanial resistance.

Automatic cell cloning assay for determining the clonogenic capacity of cancer and cancer stem‐like cells

The clonogenic assay is a well-established in vitro method for testing the survival and proliferative capability of cells. It can be used to determine the cytotoxic effects of various treatments including chemotherapeutics and ionizing radiation. However, this approach can also characterize cells with different phenotypes and biological properties, such as stem cells or cancer stem cells. In this study, we implemented a faster and more precise method for assessing the cloning efficiency of cancer stem-like cells that were characterized and separated using a high-speed cell sorter. Cell plating onto a microplate using an automatic cell deposition unit was performed in a single-cell or dilution rank mode by the fluorescence-activated cell sorting method. We tested the new automatic cell-cloning assay (ACCA) on selected cancer cell lines and compared it with the manual approach. The obtained results were also compared with the results of the limiting dilution assay for different cell lines. We applied the ACCA to analyze the cloning capacity of different subpopulations of prostate and colon cancer cells based on the expression of the characteristic markers of stem (CD44 and CD133) and cancer stem cells (TROP-2, CD49f, and CD44). Our results revealed that the novel ACCA is a straightforward approach for determining the clonogenic capacity of cancer stem-like cells identified in both cell lines and patient samples.

‘Familial‘ Chronic Lymphocytic Leukemia

Chris Pepper speaks to Roshaine Gunawardana, Managing Commissioning Editor: Dr Chris Pepper obtained his PhD in Medicinal Chemistry from the Welsh School of Pharmacy (Cardiff, Wales, UK) in 1993. He completed his postdoctoral training in the Department of Pathology at Cardiff University, School of Medicine (Cardiff, Wales, UK) before moving to Llandough Hospital (South Glamorgan, Wales, UK) as a Clinical Scientist in Hematology. He is currently a Reader in Hematology in the Institute of Cancer &amp; Genetics in Cardiff where he runs a successful research team and provides the high-speed cell sorting facility for the University. For the past 18 years, his research career has been focused on chronic lymphocytic leukemia. During this time, he has published over 80 peer-reviewed papers and has been an invited speaker at numerous national and international meetings. His research team is currently addressing basic, translational and clinical questions in chronic lymphocytic leukemia. These include investigations into the mechanisms that underpin the development of disease progression and drug resistance, elucidation of novel biomarkers of prognosis in order to provide superior risk stratification for individual patients, and the development of novel targeted therapeutic agents.

High-speed cell sorter

High-speed cell sorter

Isolation of synaptic terminals from Alzheimer's disease cortex

Amyloid beta (Aβ) oligomers and phosphorylated tau (p-tau) aggregates are increasingly identified as potential toxic intermediates in Alzheimer's disease (AD). In cortical AD synapses, p-tau co-localizes with Aβ, but the Aβ and p-tau peptide species responsible for synaptic dysfunction and demise remains unclear. The present experiments were designed to use high-speed cell sorting techniques to purify synaptosome population based on size, and then extend the method to physically isolate Aβ-positive synaptosomes with the goal of understanding the nature of Aβ and tau pathology in AD synapses. To examine the purity of size-gated synaptosomes, samples were first gated on size; particles with sizes between 0.5 and 1.5 microns were collected. Electron microscopy documented a homogenous population of spherical particles with internal vesicles and synaptic densities. Next, size-gated synaptosomes positive for Aβ were collected by fluorescence activated sorting and then analyzed by immunoblotting techniques. Sorted Aβ-positive synaptosomes were enriched for amyloid precursor protein (APP) and for Aβ oligomers and aggregates; immunolabeling for p-tau showed a striking accumulation of p-tau aggregates compared to the original homogenate and purified synaptosomes. These results confirm co-localization of Aβ and p-tau within individual synaptic terminals and provide proof of concept for the utility of flow sorting synaptosomes.

Acute Bilineal Leukemia Is a Very Rare Entity in Childhood

Abstract 4871Recent WHO 2008 classification introduced a new category named Mixed Phenotype Acute Leukemia (MPAL) for leukemias in which primary lineage cannot be determined by morphology, cytochemistry and/or flow cytometry (FC). Acute bilineal leukemia (ABL) is a subtype of MPAL and is defined by presence of distinct myeloid and lymphoid clonal populations simultaneously at diagnosis. No epidemiological data on ABL have been published so far and there are also few data on the origin of distinct leukemic clones. We examined the incidence and biology of ABL cases among children with primary acute leukemia in the period between 1996 and 2011 in the Czech Republic. Morphology and FC were centrally evaluated in all patients. In total 1065 patients were diagnosed (919 ALL, 146 AML); out of them 3 patients were classified as ABL. Two cases had simultaneous presence of distinct B-cell precursor (BCP) and myeloid clones (BCP-My) at diagnosis, one patient had discrete T ALL and myeloid population (T-My). All ABL patients were screened for immunoglobulin (Ig) and T-cell receptor (TCR) clonality, BCR-ABL, MLL gene rearrangements and FLT3-ITD. All three patients had detectable clonality in lymphoid-specific Ig/TCR rearrangements. In pt1 (BCP-My), FLT3-ITD abnormality and in pt3 (BCP-My), BCR-ABL fusion gene were found. Both patients with BCP-My ABL had Ikaros (IKZF1) gene deletion. In pt2 complex karyotype with MLL gene translocation was identified. Using high speed cell sorting we evaluated the presence or absence of previously mentioned changes in separated subpopulations. In patients with BCP-My ABL, identical clonal Ig rearrangements were found in both lymphoid and myeloid clones. Also FLT3-ITD and BCR-ABL aberrations were present in both clones of respective patients. In pt2 (T-My) TCR gene rearrangement was absent in myeloid population. All three patients achieved complete remission (CR) by lymphoid-directed induction treatment followed by switch to myeloid-oriented blocks according Interfant 99, resp. 2006 protocol in pt 1 and 2 and ALL treatment combined with tyrosine kinase inhibitor (TKI) in pt3. Finally all patients underwent allogeneic hematopoietic stem cell transplantation (SCT) in first CR. Pt1 relapsed after SCT as “typical” cALL. However, the plasticity was maintained and the myeloid clone reappeared at day 28 of relapse therapy. Pt2 relapsed as AML with undetectable TCR gene rearrangements. Pt3 is in complete remission 23 months after SCT with detectable low-level MRD and mixed chimerism with repeated lowering MRD after re-administered TKI dasatinib. Conclusions:ABL is an extremely rare entity in childhood accounting for less than 0.3 % of all acute leukemia cases. Surprisingly, the same genetic changes can be identified in both clonal populations making the “true” ABL even rarer. FC in combination with morphology is the basic method for identifying ABL and should be followed by detailed genetic analysis. The prognosis of ABL patients in our cohort was poor, despite SCT preceded by the application of treatment modalities targeting both lymphoid and myeloid clones. Disclosures:No relevant conflicts of interest to declare.

Improvement of Particle Alignment Control and Precise Image Acquisition for On-Chip High-Speed Imaging Cell Sorter

Improvement of Particle Alignment Control and Precise Image Acquisition for On-Chip High-Speed Imaging Cell Sorter

Improvement of Particle Alignment Control and Precise Image Acquisition for On-Chip High-Speed Imaging Cell Sorter

We have developed a real-time imaging cell sorting system composed of a micrometer-sized gel-electrode-embedded microfluidic sorting chip and a real-time image analysis/recognition unit equipped with a high-speed camera and image processing circuits. For the microfluidic continuous cell sorting, we have examined the precise position and velocity control of flowing particles and the precise acquisition of microscopic images of flowing particles. The results showed that (1) hydrodynamic focusing can line up particles precisely within a range of 5 µm particle size distribution, (2) active air pressure-driven flow velocity control can create the flow in the microfluidic pathways up to 160 mm/s with 0.15 MPa air pressure maintaining linear correlation between air pressure and flow velocity, and (3) 1 µs flash illumination can prevent the blur even under 200 mm/s flow. Applying the above elements into the system, the recognition error of target particles was within 5% for 2 µm particles with 2.5 mm/s flow. The experimental results demonstrate the potential of the image index-based on-chip cell sorter for practical application.

Flow cytometric sorting of the filamentous fungus Trichoderma reesei for improved strains

Metabolic measurements and screening of Trichoderma reesei have conventionally been performed during the hyphal stage of fungal development. To determine if flow cytometric measurements of protein expression could be made on germinating spores we created a gene construct, placing the Renilla reniformis green fluorescent protein gene under control of the cellobiohydrolase I ( cbh1) promoter and terminator of T. reesei. This vector was transformed into T. reesei and GFP expression was measured in germlings by flow cytometry. Fluorescence associated with GFP expression was observed in germlings grown under conditions known to induce cellulases in Trichoderma. Spores were mutated using UV light and germinating spores were screened for increased GFP expression using high-speed cell sorting, to select for strains with genetic changes associated with increased protein expression. Secondary screens for cellulase production were conducted in microtitre plates. Flow cytometric screening of germinating spores expressing GFP yielded a mutant with improved ability to hydrolyse biomass.

Abstract LB-242: Oncosomes: Large bioactive particles secreted by prostate cancer cells

Abstract Purpose of the study: Human prostate tumor cells were recently shown by our group to secrete large (0.5-10 m diameter) bioactive microvesicles capable of activating signal transduction and inducing proliferation and migration in cells in the tumor microenvironment (Di Vizio, et al. Cancer Res. 2009 69: 5601-9). These secreted particles, termed oncosomes, were shown to be vehicles for numerous signaling proteins involved in cell growth and motility. They also contain the membrane protein caveolin-1 (Cav-1), a mediator of prostate cancer (PCa) metastasis and a circulating PCa biomarker. The purpose of this study was to develop the tools to identify, isolate, sort and characterize PCa-derived oncosomes. Experimental procedures: Secreted particles from PCa cells were ultracentrifuged at 100,000xg and the pelleted material processed on a Moflo High-Speed Cell Sorter (Beckman-Coulter) and analyzed using Summit V4.3 software (Dako Colorado, Inc.). 1, 3 and 10 m bead standards (Spherotech Inc.) were used to set size gates. To avoid capturing aggregated material, forward scatter and pulse width were set at a linear scale and only single events were gated and analyzed. Sorted particles were visualized by confocal and electron microscopy. Results: Using the above procedure, we were able to retrieve intact particles of 1-10 m in diameter secreted by LNCaP cells overexpressing the oncoprotein MyrAkt1. The nontumorigenic prostate stromal cell line WPMY-1 secreted &amp;gt; 10-fold fewer particles in this size range. We confirmed that these were membrane-derived particles by showing that they contained MyrAkt1, which localizes to the plasma membrane. Particles in a similar size range were also sorted after purification from LNCaP cells stably expressing a Cav-1/GFP fusion protein. Sorted particles visualized microscopically appeared to be surrounded by a lipid bilayer and to have intrinsic structure that made them resistant to collapse. Particles isolated by these methods evoked degradation of FITC-labeled collagen and contained active MMP9 and MMP2, two key proteases involved in tumor cell invasion. Conclusions: The specific type of large bioactive particle we have isolated has not been previously described in any tumor system. Studies are ongoing to detect these particles in animal models of cancer and in human patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-242.

Augmented Revascularization and Recovery From Acute Limb Ischemia After Transplantation of Human Umbilical Cord Blood Progenitor Cells with High Aldehyde Dehydrogenase Activity.

Abstract 3042Poster Board II-1018Clinical application of regenerative cell-based therapies for the treatment of ischemic vascular disease has proven challenging to implement due the involvement of multiple human cell types that co-ordinate angiogenesis. Previously, transplanted human progenitor cells from hematopoietic, endothelial, and mesenchymal lineages have all been implicated in the recovery and de novo production of perfused blood vessels in vivo. Using high aldehyde dehydrogenase (ALDHhi) activity, a conserved function of these pro-angiogenic hematopoietic and non-hematopoietic progenitor cell lineages, we have recently shown that transplanted human bone marrow (BM) ALDHhi cells transiently recruit to areas of hypoxia and augment revascularization and recovery of perfusion in ischemic limbs via the stimulation of endogenous vascular regeneration. However, clinical use of autologous BM-derived cells in patients with critical limb ischemia may be limited by the availability or dysfunction of transplanted pro-angiogenic cells due to vascular disease-related pathologies. The non-invasive collection and early ontogeny of human umbilical cord blood (UCB) progenitor cells provides a promising alternative source of pro-angiogenic progenitor cells with vascular regenerative functions. Human UCB mononuclear cells (MNC) were purified using high-speed fluorescence-activated cell sorting to accrue clinically applicable cell populations with low side scatter and high (ALDHhi, 0.4±0.1% of total MNC) versus low (ALDHlo, 41.2±3.6% of total MNC) ALDH activity (n=10). Compared to UCB-derived ALDHlo cells, which were devoid of hematopoietic and non-hematopoietic progenitor function in vitro, ALDHhi cells were enriched for hematopoietic colony forming cells (1 HCFC in 3.6 cells, n=4) and endothelial colony forming cells (1 ECFC in 5.8×104 cells, n=4) enumerated after 14 days in culture. In contrast to human BM-derived ALDH-purified cells which were enriched for mesenchymal colony forming cells, analogous ALDHlo and ALDHhi cells from UCB did not establish mesenchymal-stromal colonies in vitro suggesting a comparative deficiency in mesenchymal stem cell frequency after UCB venipuncture. We investigated the vascular regenerative capacity of these human UCB-derived ALDHlo and ALDHhi cells by tail-vein transplantation into sublethally irradiated (275cGy) immune-deficient, β-glucuronidase (GUSB) deficient, NOD/SCID/MPSVII mice with acute limb ischemia induced by unilateral femoral artery ligation and transection. Using weekly laser Doppler perfusion imaging to track the kinetics of blood flow recovery indicated by the perfusion ratio (PR) in the ischemic versus non-ischemic limb, mice transplanted with 2×105-4×105 purified ALDHhi cells showed significantly improved recovery of limb perfusion by day 21 post-transplantation (PR=0.70±0.06, n=6) compared to mice injected with phosphate-buffered saline (PR=0.35±0.06, n=5, P<0.01), 10×106 ALDHlo cells (PR=0.45±0.07, n=7, P<0.01), or 20×106 unpurified UCB MNC (PR=0.40±0.06, n=7, P<0.01). Furthermore, transplantation of ALDHhi progenitor cells augmented revascularization in the ischemic limb compared to all other treatments at day 28 post-transplantation (P<0.01), quantified by immunohistochemical detection and enumeration of von Willebrand factor+ blood vessels in frozen sections from the ischemic adductor muscle. Only mice transplanted with ALDHhi cells showed retention of human hematopoietic (CD45+/HLA-A,B,C+) cells in the ischemic adductor muscle for 28 days (3.0±2.1%, n=6) quantified by flow cytometry. Ischemic muscle sections from NOD/SCID/MPSVII mice transplanted with human UCB ALDHhi cells confirmed human cell retention and engraftment adjacent to damaged vasculature and ischemic muscle fibres at day 28 post-transplantation, measured by colorimetric detection of ubiquitous GUSB activity in transplanted human cells. Human UCB-derived ALDHhi cells, which were devoid of mesenchymal-stromal cells but enriched for potentially pro-angiogenic endothelial and hematopoietic progenitor lineages, showed persistent retention in areas of hypoxia and augmented the endogenous revascularization and recovery of perfusion in ischemic limbs. Thus, ALDHhi mixed progenitor cells may prove to be a useful allogeneic alternative to human BM in the clinical treatment of patients with severe peripheral vascular disease. DisclosuresNo relevant conflicts of interest to declare.

High‐Speed Cell Sorting of Infectious Trophic and Cystic Forms of Pneumocystis carinii

The separation of Pneumocystis carinii life-cycle stages while preserving infectivity is a hitherto unresolved challenge. We describe an original, reproducible, and efficient method for separating trophic from cystic forms of P. carinii using a high-speed cell sorter. The large amounts of highly purified (99.6+/-0.3%) infectious trophic and cystic forms can now be used to elucidate the poorly understood P. carinii life cycle.

Amplification of Porcine SRY Gene for Sex Determination

The separation of X and Y chromosome-bearing sperm is of use in many aspects of livestock maintenance. In this study, we sought to determine the difference in DNA content between X- and Y-bearing sperm, separate sperm into X- and Y-enriched pools, and assess the efficacy of sorting. Sperm collected from Duroc and miniature pigs were stained with 20.8 μM Hoechst 33342 and analyzed using a high-speed cell sorter. Measurement of the fluorescence intensity of stained sperm nuclei revealed that the X-bearing sperm of Duroc and miniature pigs respectively contain 2.75% and 2.88% more DNA than Y-bearing sperm. In total, 50.18% of the sperm were assigned to the X-sorted sample and 49.82% was assigned to the Y-sorted sample for Duroc pigs. For miniature pigs, the X- sorted sample represented 50.19% of the population and the Y-sorted represented 49.81% of the population. Duplex PCR was used to evaluate accuracy of sorting. A fast and reliable method for porcine sexing was developed through amplification of the sex-determining region of the Y chromosome gene (SRY). Oligonucleotide primers were designed to amplify the conserved porcine SRY high motility group (HMG) box sequence motif. We found that the primer pair designed in this study was 1.46 times more specific than previously reported primers. Thus, this study shows that the present method can be applied in porcine breeding programs to facilitate manipulation of the sex ratio of offspring and to achieve precise sexing of porcine offspring by amplification of the HMG box of the SRY gene. (Key

Flow cytometry applications in the food industry

Flow cytometry has become a valuable tool in food microbiology. By analysing large numbers of cells individually using light-scattering and fluorescence measurements, this technique reveals both cellular characteristics and the levels of cellular components. Flow cytometry has been developed to rapidly enumerate microorganisms; to distinguish between viable, metabolically active and dead cells, which is of great importance in food development and food spoilage; and to detect specific pathogenic microorganisms by conjugating antibodies with fluorochromes, which is of great use in the food industry. In addition, high-speed multiparametric data acquisition, analysis and cell sorting, which allow other characteristics of individual cells to be studied, have increased the interest of food microbiologists in this technique. This mini-review gives an overview of the principles of flow cytometry and examples of the application of this technique in the food industry.

Successful low dose insemination of flow cytometrically sorted Sika ( Cervus nippon) sperm in Wapiti ( Cervus elaphus)

The purpose of this study was to determine a practical method in Wapiti ( Cervus elaphus) of using predetermined sexed Sika ( Cervus nippon) semen. Semen was collected by electro-ejaculation from one stag of proven fertility and transported to the laboratory where it was retained as unsorted (control) or was separated into X- and Y-chromosome-bearing sperm using a modified high-speed cell sorter. Wapiti hinds ( n = 81) were inseminated into the uterus by rectum manipulation with 1 × 10 6 (X1 and Y1 group, respectively) or 2 × 10 6 (X2 and Y2 group, respectively) of sorted frozen-thawed and 1 × 10 7 non-sorted frozen-thawed (a commercial dose control) Sika motile sperm 60–66 h after removal of intra-vaginal progesterone-impregnated CIDR devices and administration of 700 IU of PMSG at the time of CIDR removal. The percentage of hinds calving after insemination was similar for X1 (38.5%), X2 (41.7%), Y1 (44.4%), Y2 (38.9%) groups ( P > 0.05), but higher for control (75%) treatment ( P < 0.05). Ultimately 15 out of the 16 Sika and Wapiti-hybrid calves produced by Wapiti hinds inseminated with Y-sorted sperm were male (93.7%) and 10/10 (100%) Sika and Wapiti-hybrid calves from hinds inseminated with X-sorted sperm were female. The sex ratio of the Sika and Wapiti-hybrid calves born to hinds inseminated with sex-sorted sperm deviated significantly ( P < 0.05) from 50% and 50.0% in the control group. All Sika and Wapiti-hybrid calves were born between 237 and 250 d of gestation. Male and female calves in the control group had similar birth weights and weaning weights as calves from hinds inseminated with X- or Y-sorted sperm. In conclusion it can be said that normal Sika and Wapiti-hybrid calves of predicted sex can be produced after artificial insemination of Wapiti does with low numbers of sex-sorted cryopreserved Sika sperm.