Discontinuous Density Gradient Centrifugation Research Articles

Quantification of X sperm by raman spectroscopy in percoll density gradient centrifuged buffalo semen

The present study was conducted to observe effect of percoll density gradient centrifugation of buffalo bull semen on quantity of X sperms. Ejaculates were collected by artificial vagina method. Semen with mass motility >+3 and progressive motility >70 % were selected for experiment. X sperm Enrichment of semen was done by discontinuous percoll density gradient centrifugation and three groups were formed ie Group 1 (3 layer 70%, 50% and 30%) Group 2 (7 layer 70%, 60%, 50%, 40%, 30%, 20% and 10%) Group 3 (7 layer 80%, 70%, 60%, 50%, 40%, 30% and 20%). Centrifugation of semen of three groups and control (fresh semen without gradients) was done. After centrifugation, the supernatant part was removed and the pellet of each group was used for X sperm enrichment assessment by Raman spectroscopy. Results revealed that X sperm enrichment was higher in the pellets of Group 2 followed Group 3, Group 1 and Control as Raman peaks on DNA specific bands corresponds to more number of x sperm were higher respectively.

CD41+ extracellular vesicles produced by avian thrombocytes contain microRNAs.

Avians have thrombocytes in their blood circulation rather than mammalian platelets. However, many details of thrombocyte characteristics have not been determined. Here, chicken thrombocytes were isolated, and extracellular vesicle (EV) production was investigated. The thrombocyte-specific markers cd41 and cd61 were expressed in the yolk sac at 24 h. According to the embryonic developmental stage, the cd41-expressing tissues changed from the yolk sac to the bone marrow and spleen. Accordingly, the bone marrow and spleen were the main tissues producing thrombocytes in adult chickens. Avian thrombocytes were separated from adult spleen cells through a combination of discontinuous density gradient centrifugation, phagocytic cell removal, and fluorescence-activated cell sorting. Isolated thrombocytes produced CD41+ EVs (CD41+ EVs), and the CD41+ EVs also expressed CD9. Microarray analysis revealed that CD41+ EVs contain many microRNAs. Macrophage lines (RAW264.7) phagocytosed CD41+ EVs, and their phagocytosis and migration activity were suppressed. Microarray analysis also revealed that EVs altered gene expression in macrophages. These data indicated that the CD41+ EV was a carrier of microRNAs produced from thrombocytes and affected the cell characteristics of the received cells. Therefore, the CD41+ EVs of avians worked as a communication tool.

Nano-Sized Chimeric Human Papillomavirus-16 L1 Virus-like Particles Displaying Mycobacterium tuberculosis Antigen Ag85B Enhance Ag85B-Specific Immune Responses in Female C57BL/c Mice.

Bacillus Calmette-Guerin (BCG), the only current vaccine against tuberculosis (TB) that is licensed in clinics, successfully protects infants and young children against several TB types, such as TB meningitis and miliary TB, but it is ineffective in protecting adolescents and adults against pulmonary TB. Thus, it is a matter of the utmost urgency to develop an improved and efficient TB vaccine. In this milieu, virus-like particles (VLPs) exhibit excellent characteristics in the field of vaccine development due to their numerous characteristics, including but not limited to their good safety without the risk of infection, their ability to mimic the size and structure of original viruses, and their ability to display foreign antigens on their surface to enhance the immune response. In this study, the HPV16 L1 capsid protein (HPV16L1) acted as a structural vaccine scaffold, and the extracellular domain of Ag85B was selected as the M. tb immunogen and inserted into the FG loop of the HPV16 L1 protein to construct chimeric HPV16L1/Ag85B VLPs. The chimeric HPV16L1/Ag85B VLPs were produced via the Pichia pastoris expression system and purified via discontinuous Optiprep density gradient centrifugation. The humoral and T cell-mediated immune response induced by the chimeric HPV16L1/Ag85B VLP was studied in female C57BL/c mice. We demonstrated that the insertion of the extracellular domain of Ag85B into the FG loop of HPV16L1 did not affect the in vitro stability and self-assembly of the chimeric HPV16L1/Ag85B VLPs. Importantly, it did not interfere with the immunogenicity of Ag85B. We observed that the chimeric HPV16L1/Ag85B VLPs induced higher Ag85B-specific antibody responses and elicited significant Ag85B-specific T cell immune responses in female C57BL/c mice compared with recombinant Ag85B. Our findings provide new insights into the development of novel chimeric HPV16L1/TB VLP-based vaccine platforms for controlling TB infection, which are urgently required in low-income and developing countries.

Percoll discontinuous density gradient centrifugation method for the fractionation of the subpopulations of Mycobacterium smegmatis and Mycobacterium tuberculosis from in vitro cultures

Bacterial populations in the in vitro laboratory cultures, environment, and patients contain metabolically different subpopulations that respond differently to stress agents, including antibiotics, and emerge as stress tolerant or resistant strains. To contain the emergence of such strains, it is important to study the features of the metabolic status and response of the subpopulations to stress agents. For this purpose, an efficient method is required for the fractionation and isolation of the subpopulations from the cultures. Here we describe in detail the manual setting up of a simple, easy-to-do, reproducibly robust Percoll discontinuous density gradient centrifugation for the fractionation of subpopulations of short-sized cells (SCs) and normal/long-sized cells (NCs) from Mycobacterium smegmatis and Mycobacterium tuberculosis cultures, which we had reported earlier. About 90-98% enrichment was obtained respectively for SCs and NCs for M. smegmatis and 69-67% enrichment was obtained respectively for the SCs and NCs for M. tuberculosis.•The Percoll discontinuous density gradient centrifugation helps the fractionation and isolation of mycobacterial subpopulations that differ in density.•The method offers a consistently reproducible high enrichment of the subpopulations of SCs and NCs from the in vitro cultures of M. smegmatis and M. tuberculosis.•Our earlier reports on the consistency in the differential response of the subpopulations, enriched using the method, to oxidative, nitrite, and antibiotic stress proves its validity.

Determination of short- and medium-chain chlorinated paraffins in different components of human blood using gas chromatography-electron capture negative ion-low resolution mass spectrometry

Short- and medium-chain chlorinated paraffins (SCCPs and MCCPs) have attracted significant attention because of their persistence, biotoxicity, bioaccumulation, and long-range migration. Given their worldwide detection in a variety of environmental matrices, concerns related to the high exposure risks of SCCPs and MCCPs to humans have grown. Thus, knowledge of the contamination patterns of SCCPs and MCCPs and their distribution characteristics in the vivo exposure of humans is of great importance. However, little information is available on the contamination of SCCPs and MCCPs in human blood/plasma/serum, mainly because of the difficulty of sample preparation and quantitative analysis. In this study, a new blood sample pretreatment method based on Percoll discontinuous density gradient centrifugation was developed to separate plasma, red blood cells, white blood cells, and platelets from human whole blood. A series of Percoll sodium chloride buffer solutions with mass concentrations of 1.095, 1.077, and 1.060 g/mL were placed in a centrifuge tube from top to bottom to establish discontinuous density gradients. The dosage for each density gradient was 1.5 mL. Human whole blood samples mixed with 0.85% sodium chloride aqueous solution were then added to the top layer of the Percoll sodium chloride solution. After centrifugation, the whole blood was separated into four components. The plasma was located at the top layer of the centrifuge tube, whereas the platelets, white blood cells, and red blood cells were retained at the junction of the various Percoll sodium chloride solutions. The sampling volume of human whole blood and incubation time were optimized, and results indicated that an excessively long incubation time could lead to hemolysis, resulting in a decrease in the recoveries of SCCPs and MCCPs. Therefore, a sampling volume of 1.5 mL and incubation time of 10 min at 4 ℃ were adopted. The cells of the blood components were further broken and extracted by ultrasonic pretreatment, followed by multilayer silica gel column chromatography for lipid removal. The use of 80 mL of n-hexane-dichloromethane (1∶1, v/v) and 50 mL of dichloromethane as the elution solvents (collected together) for the gel column separated the SCCPs and MCCPs from the lipid molecules in the blood samples. Gas chromatography-electron capture negative ion-low resolution mass spectrometry (GC-ECNI-LRMS) was used to determine the SCCPs and MCCPs. Quantification using the corrected total response factor with degrees of chlorination was achieved with linear corrections (R2=0.912 and 0.929 for the SCCPs and MCCPs, respectively). The method detection limits (MDLs) for the SCCPs and MCCPs were 1.57 and 8.29 ng/g wet weight (ww, n=7), respectively. The extraction internal standard recoveries were 67.0%-126.6% for the SCCPs and 69.5%-120.5% for the MCCPs. The developed method was applied to determine SCCPs and MCCPs in actual human whole blood samples. The contents of SCCPs and MCCPs were 10.81-65.23 and 31.82-105.65 ng/g (ww), respectively. Red blood cells exhibited the highest contents of CPs, followed by plasma, white blood cells, and platelets. The proportions of SCCPs and MCCPs in red blood cells and plasma were 70% and 66%, respectively. In all four components, the MCCP contents were higher than the SCCP contents, and the ratios of MCCPs to SCCPs ranged from 1.04 to 3.78. Similar congener patterns of SCCPs and MCCPs were found in the four components of human whole blood. C10-CPs and C14-CPs were predominantly observed in the SCCPs and MCCPs, respectively. In summary, a simple and efficient method was proposed to determine low concentrations of SCCPs and MCCPs in human blood with high sensitivity and selectivity. This method can meet requirements for the quantitative analysis of SCCPs and MCCPs in human blood components, thereby providing technical support for human health risk assessment.

P-059 Sperm DNA fragmentation (SDF) after cryopreservation and sperm selection: implications for clinical pregnancies and live births after intrauterine insemination with donor sperm

Abstract Study question Can SDF assessment pre-cryopreservation, post-thaw and after post-thaw sperm selection predict clinical outcome in a heterologous intra-uterine donor insemination program (IUI)? Summary answer SDF increased post-cryopreservation in donor sperm and after post-thaw density gradient without affecting clinical pregnancy, live birth and miscarriage rates. What is known already Sperm cryopreservation is effective for preservation of male fertility and facilitates the storage of donor semen, while infectious disease screening can be completed and confirmed negative. Sperm viability and motility are the most vulnerable parameters during the freeze-thaw process. Inherent reactive oxygen species may induce SDF via oxidative stress. Controversial results are reported regarding induction of SDF after cryopreservation due to differences in cryo tolerance, methods of cryopreservation and methods used to evaluate SDF. Sperm selection post-thaw is indispensable to select a normal motile fraction for insemination. Again, results are controversial regarding influence on SDF levels after sperm selection post-thaw. Study design, size, duration This was a prospective, observational study. Between March 2015 and March 2019, eighteen potential sperm donors were screened via a three step plan – semen assessment, medical assessment and serological blood tests. In accordance with the Belgian legislation (2007), a sperm donor was matched to obtain a maximum of 6 pregnancies in acceptor women/couples, allowing for more than one offspring/woman or couple. A total of 106 acceptors were matched for heterologous intra-uterine insemination. Participants/materials, setting, methods Semen samples were collected after 2-7 days abstinence and standard semen parameters analyzed within 60 mins after ejaculation. Within the same time frame SDF test was performed using TUNEL assay both before and after cryopreservation and after a two-step discontinuous density gradient centrifugation post-thaw. Samples were cryopreserved by the conventional slow freezing method using sperm freeze solution. A yield of ≥ 2 M progressive spermatozoa post-thaw after selection was sufficient to contemplate IUI. Main results and the role of chance Mean female age at first cycle was 33.9 ± 4.1 years and donor age 28.5 ± 5.6 years. Out of 429 cycles, 100 (23.3%) resulted in clinical pregnancy. Excluding three ongoing pregnancies, we counted 75 live births (17.6% of cycles or 77.3% of pregnancies), while 21 pregnancies ended in miscarriage (4.9% of cycles or 21.6% of pregnancies) and 1 resulted in stillbirth. Progressive motility (59.3 ± 12.5%) decreased significantly after cryopreservation (30.0 ± 13.2%; p < 0.001), but increased post-thaw after density gradient centrifugation (61.6 ± 16.0%; p < 0.001). On the contrary, SDF (12.0 ± 5.9%) increased post-cryopreservation (26.3 ± 14.5%; p < 0.001) and further increased after sperm selection (34.9 ± 22.1%; p = 0.04). Using multilevel mixed-effects logistic regression (Odds ratio [95%CI]), we found that female age significantly influenced clinical pregnancy (0.911 [0.847-0.981]; p = 0.01), live birth (0.894 [0.834-0.959]; p = 0.002) and miscarriage rates (1.180 [1.033-1.347]; p = 0.015). There was no significant age-adjusted effect of SDF post-thaw after density gradient selection on clinical pregnancy (1.007 [0.994-1.021]; p = 0.30), live birth (1.001 [0.988-1.014]; p = 0.84) or miscarriage (1.021 [0.997-1.046]; p = 0.08). Median time-to-live birth was 4 cycles. Analyses of time-to-live birth with mixed-effects Cox models revealed no significant association with progressive motility; SDF before or after cryopreservation and after sperm selection post-thaw. Limitations, reasons for caution Results concern a small donor population, with high normal semen parameters and should therefore be extrapolated cautiously to subfertile men with normal and subnormal semen parameters. Wider implications of the findings In a donor IUI program with strict selection on conventional sperm parameters, differences in SDF before and after cryopreservation and gradient centrifugation do not change IUI outcomes, although cryopreservation and centrifugation increase SDF. Findings could be useful for the optimization of sperm freezing and selection in different assisted reproductive procedures. Trial registration number not applicable

Separation of Single Core and Multicore Lytic Granules by Subcellular Fractionation and Immunoisolation.

Subcellular fractionation is an important tool used to separate intracellular organelles, structures or proteins. Here, we describe a stepwise protocol to isolate two types of lytic granules, multicore (MCG), and single core (SCG), from primary murine CTLs. We used cell disruption by nitrogen cavitation followed by separation of organelles via discontinuous sucrose density gradient centrifugation. Immunoisolation with a Synaptobrevin 2 antibody attached to magnetic beads was then used to harvest Synaptobrevin 2 positive granules for immunoblotting, mass spectrometry, electron, and light microscopy.

A method for isolating highly purified and active mitochondria from insects

So far, methods that yield the high purity and activity of the isolated mitochondria from insects have not been reported and determined. Here, we develop methods that combine differential centrifugation and discontinuous Nycodenz density gradient centrifugation to isolate highly purified mitochondria from the thorax muscle of insects, and the methods were widely validated across three orders (Coleoptera, Hymenoptera, and Blattaria) covering four insect species using Western blot and transmission electron microscopy (TEM) analysis. The results showed the removal of the residual contamination with nonmitochondrial components such as nucleus, sarcolemma, cytosol, and endoplasmic reticulum. Furthermore, TEM, mitochondria staining, fluorescence detection, and flow cytometry analyses were employed to assess membrane integrity and activity of the isolated mitochondria. The results showed no loss of mitochondria activity/integrity after isolation. In addition, temporal dynamics in activity of the isolated mitochondria under commonly used laboratory temperature (-20 °C, 4 °C, and 25 °C) were respectively detected using a fluorescence microplate reader. The results showed that it should be avoided to store the isolated mitochondria at room temperature, and the mitochondria can meet the requirements of the most downstream experiments when they were stored at −20 °C. Overall, the study presented a method for isolating highly purified and active mitochondria from insects. This study firstly described a high-speed discontinuous density gradient centrifugation-based method that could be widely applied for mitochondria isolation in insects. The present study also provided an example to assess purity and integrity/activity of the isolated mitochondria.

Effect of High-Density Lipoprotein from Healthy Subjects and Chronic Kidney Disease Patients on the CD14 Expression on Polymorphonuclear Leukocytes.

In uremic patients, high-density lipoprotein (HDL) loses its anti-inflammatory features and can even become pro-inflammatory due to an altered protein composition. In chronic kidney disease (CKD), impaired functions of polymorphonuclear leukocytes (PMNLs) contribute to inflammation and an increased risk of cardiovascular disease. This study investigated the effect of HDL from CKD and hemodialysis (HD) patients on the CD14 expression on PMNLs. HDL was isolated using a one-step density gradient centrifugation. Isolation of PMNLs was carried out by discontinuous Ficoll-Hypaque density gradient centrifugation. CD14 surface expression was quantified by flow cytometry. The activity of the small GTPase Rac1 was determined by means of an activation pull-down assay. HDL increased the CD14 surface expression on PMNLs. This effect was more pronounced for HDL isolated from uremic patients. The acute phase protein serum amyloid A (SAA) caused higher CD14 expression, while SAA as part of an HDL particle did not. Lipid raft disruption with methyl-β-cyclodextrin led to a reduced CD14 expression in the absence and presence of HDL. HDL from healthy subjects but not from HD patients decreased the activity of Rac1. Considering the known anti-inflammatory effects of HDL, the finding that even HDL from healthy subjects increased the CD14 expression was unexpected. The pathophysiological relevance of this result needs further investigation.

Dual-process brain mitochondria isolation preserves function and clarifies protein composition

The brain requires continuously high energy production to maintain ion gradients and normal function. Mitochondria critically undergird brain energetics, and mitochondrial abnormalities feature prominently in neuropsychiatric disease. However, many unique aspects of brain mitochondria composition and function are poorly understood. Developing improved neuroprotective therapeutics thus requires more comprehensively understanding brain mitochondria, including accurately delineating protein composition and channel-transporter functional networks. However, obtaining pure mitochondria from the brain is especially challenging due to its distinctive lipid and cell structure properties. As a result, conflicting reports on protein localization to brain mitochondria abound. Here we illustrate this problem with the neuropsychiatric disease-associated L-type calcium channel Cav1.2α1 subunit previously observed in crude mitochondria. We applied a dual-process approach to obtain functionally intact versus compositionally pure brain mitochondria. One branch utilizes discontinuous density gradient centrifugation to isolate semipure mitochondria suitable for functional assays but unsuitable for protein localization because of endoplasmic reticulum (ER) contamination. The other branch utilizes self-forming density gradient ultracentrifugation to remove ER and yield ultrapure mitochondria that are suitable for investigating protein localization but functionally compromised. Through this process, we evaluated brain mitochondria protein content and observed the absence of Cav1.2α1 and other previously reported mitochondrial proteins, including the NMDA receptor, ryanodine receptor 1, monocarboxylate transporter 1, excitatory amino acid transporter 1, and glyceraldehyde 3-phosphate dehydrogenase. Conversely, we confirmed mitochondrial localization of several plasma membrane proteins previously reported to also localize to mitochondria. We expect this dual-process isolation procedure will enhance understanding of brain mitochondria in both health and disease.

Isolation and comparative proteomic analysis of mitochondria from the pulp of ripening citrus fruit

Mitochondria are crucial for the production of primary and secondary metabolites, which largely determine the quality of fruit. However, a method for isolating high-quality mitochondria is currently not available in citrus fruit, preventing high-throughput characterization of mitochondrial functions. Here, based on differential and discontinuous Percoll density gradient centrifugation, we devised a universal protocol for isolating mitochondria from the pulp of four major citrus species, including satsuma mandarin, ponkan mandarin, sweet orange, and pummelo. Western blot analysis and microscopy confirmed the high purity and intactness of the isolated mitochondria. By using this protocol coupled with a label-free proteomic approach, a total of 3353 nonredundant proteins were identified. Comparison of the four mitochondrial proteomes revealed that the proteins commonly detected in all proteomes participate in several typical metabolic pathways (such as tricarboxylic acid cycle, pyruvate metabolism, and oxidative phosphorylation) and pathways closely related to fruit quality (such as γ-aminobutyric acid (GABA) shunt, ascorbate metabolism, and biosynthesis of secondary metabolites). In addition, differentially abundant proteins (DAPs) between different types of species were also identified; these were found to be mainly involved in fatty acid and amino acid metabolism and were further confirmed to be localized to the mitochondria by subcellular localization analysis. In summary, the proposed protocol for the isolation of highly pure mitochondria from different citrus fruits may be used to obtain high-coverage mitochondrial proteomes, which can help to establish the association between mitochondrial metabolism and fruit storability or quality characteristics of different species and lay the foundation for discovering novel functions of mitochondria in plants.

Diagnostic performance of discontinuous density gradient centrifugation for estimating human semen quality.

Semen analysis plays an important role in the diagnosis of male infertility. However, many studies have demonstrated that the current methods of semen analysis are inefficient for assessing male fertility. To test whether prior discontinuous density gradient centrifugation (DDCG) improves the performance of semen analysis in diagnosing male infertility. Infertile men and fertile men were recruited from the clinic. Pre- and post-DDGC values for the semen parameters of sperm concentration, total sperm number, percent total motility, percent progressive motility, percent normal sperm morphology, and sperm DNA fragmentation rate were compared. A total of 528 men (252 infertile men and 276 fertile men) were enrolled in the present study. After DDGC, sensitivity was significantly increased for sperm concentration, total sperm number, and sperm morphology (P<.01); specificity was significantly increased for progressive motility and sperm morphology (P<.01); and diagnostic accuracy was significantly improved for all of these parameters (area under the curve (AUC): P<.01). Total motility and sperm DNA fragmentation rate exhibited no obvious change in sensitivity, specificity or accuracy after DDGC (all P>.01). For the combination of all these semen parameters, diagnostic accuracy improved significantly after DDGC (AUC: P<.01). In a multiple regression analysis, only sperm morphology and sperm DNA fragmentation rate had P values less than 0.05 before DDGC, whereas all parameters except total sperm number contributed to the equation after DDGC. DDGC is a mature, standardized procedure for clinical commonly used to optimize spermatozoa. The diagnostic accuracy of semen analysis was significantly improved after DDGC, which indicated that assessing "functional spermatozoa" might be a more suitable method for semen analysis than the WHO 2010 criteria. Assessing semen parameters after DDGC might improve their diagnostic accuracy for male infertility.

Mass-Added Density Modulation for Sorting Cells Based on Differential Surface Protein Levels.

Cell sorting is a powerful tool in basic research and therapeutic enrichment. However, common cell sorting methods, such as fluorescence-activated cell sorting (FACS) and magnetic-activated cell sorting (MACS) have significant limitations, such as generally low cell yields or restriction to binary separation, respectively. To address these limitations, we developed a two-step cell sorting method called mass-added density centrifugation (MADC) to enable nonbinary separation of large cell numbers based on surface protein levels. In the first MADC step (mass-adding), antibody-directed massive microparticles bind target surface proteins to modulate single-cell density proportionally to target protein level. Second, microparticle-laden cells are subjected to discontinuous density gradient centrifugation, whereby they separate into discrete density bands which can be isolated for downstream use. MADC will prove especially advantageous for obtaining sufficient cell numbers for protein analyses from large source populations, and it is a fast process that can facilitate live cell enrichment for therapies that require tens of millions of cells. Here, we demonstrate MADC's utility for both live and fixed cell sorts of multiple cell types based on abundance of an example target protein, CD44. CD44 quantity in separated cell groups was assayed with western blots and correlated with modulated cell density. This novel sorting method enables rapid, nonbinary isolation of large quantities of cells based on surface protein levels and should prove useful in both basic science and therapeutic applications. © 2020 International Society for Advancement of Cytometry.

Comparative Efficacy of PercollTM Discontinuous Density Gradient Centrifugation and Glass Wool Filtration Techniques for Spermatozoa Selection in Buffalo (Bubalus bubalis)

Comparative Efficacy of PercollTM Discontinuous Density Gradient Centrifugation and Glass Wool Filtration Techniques for Spermatozoa Selection in Buffalo (Bubalus bubalis)

A2E Distribution in RPE Granules in Human Eyes.

A2E (N-retinylidene-N-retinylethanolamine) is a major fluorophore in the RPE (retinal pigment epithelium). To identify and characterize A2E-rich RPE lipofuscin, we fractionated RPE granules from human donor eyes into five fractions (F1–F5 in ascending order of density) by discontinuous sucrose density gradient centrifugation. The dry weight of each fraction was measured and A2E was quantified by liquid chromatography/mass spectrometry (LC/MS) using a synthetic A2E homolog as a standard. Autofluorescence emission was characterized by a customer-built spectro-fluorometer system. A significant A2E level was detected in every fraction, and the highest level was found in F1, a low-density fraction that makes up half of the total weight of all RPE granules, contains 67% of all A2E, and emits 75% of projected autofluorescence by all RPE granules. This group of RPE granules, not described previously, is therefore the most abundant RPE lipofuscin granule population. A progressive decrease in autofluorescence was observed from F2 to F4, whereas no autofluorescence emission was detected from the heavily pigmented F5. The identification of a novel and major RPE lipofuscin population could have significant implications in our understanding of A2E and lipofuscin in human RPE.

Sperm Collection of Differential Quality Using Density Gradient Centrifugation

In sexual reproduction, a male gamete or sperm cell fuses with a female gamete to bring about fertilization. However, a large number of sperm cells with fertilizing ability are required to interact with a female gamete to ensure fertilization. As such, the fertilizing ability of individual sperm cells is critical for successful reproduction. Density gradient centrifugation has been utilized for several decades as a reproducible, fast, efficient, effective and extremely adaptable method to collect only high-quality sperm to be used in assisted reproductive technology. The protocols we described herein focus on the utilization of the discontinuous Percoll density gradient centrifugation (PDGC) technique to isolate three distinct populations of rooster sperm by their quality. We were able to collect low-, medium- and high-quality sperm. We also describe reproducible protocols that entail determining fertility potential of sperm by assessing their viability, mobility and penetrability. Collection of sperm by their quality using PDGC technique would be useful to accurately and thoroughly characterize sperm with differential fertility potential.

Purification of Cyclospora cayetanensis oocysts obtained from human stool specimens for whole genome sequencing

BackgroundCyclospora cayetanensis is a food-borne intestinal human parasite that causes outbreaks of diarrhea. There is a need for efficient laboratory methods for strain-level characterization to assist in outbreak investigations. By using next generation sequencing, genomic sequences can be obtained and compared to identify potential genotyping markers. However, there is no method available to propagate this parasite in the laboratory. Therefore, genomic DNA must be extracted from oocysts purified from human stool. The objective of this study was to apply optimized methods to purify C. cayetanensis oocysts and extract DNA in order to obtain high-quality whole genome sequences with minimum contamination of DNA from other organisms.ResultsOocysts from 21 human stool specimens were separated from other stool components using discontinuous density gradient centrifugation and purified further by flow cytometry. Genomic DNA was used to construct Ovation Ultralow libraries for Illumina sequencing. MiSeq sequencing reads were taxonomically profiled for contamination, de novo assembled, and mapped to a draft genome available in GenBank to assess the quality of the resulting genomic sequences. Following all purification steps, the majority (81–99%) of sequencing reads were from C. cayetanensis. They could be assembled into draft genomes of around 45 MB in length with GC-content of 52%.ConclusionsDensity gradients performed in the presence of a detergent followed by flow cytometry sorting of oocysts yielded sufficient genomic DNA largely free from contamination and suitable for whole genome sequencing of C. cayetanensis. The methods described here will facilitate the accumulation of genomic sequences from various samples, which is a prerequisite for the development of typing tools to aid in outbreak investigations.

The immune functions of sessile hemocytes in three organs of kuruma shrimp Marsupenaeus japonicus differ from those of circulating hemocytes

Shrimp, as invertebrates, have an open vasculature that allows circulating hemocytes to infiltrate the tissues, where they are referred to as sessile hemocytes. Sessile hemocytes are known to express immune-related genes, but it is not known whether their functions differ from those of circulating hemocytes. To answer this question, we enriched them from suspensions of different tissues using discontinuous density gradient centrifugation and analyzed their transcripts by RNA-seq. The results suggest that circulating hemocytes and sessile hemocytes of the gills are in a state that could react quickly to pathogens, immune-related genes expression of sessile hemocytes differ from circulating hemocytes, and the gills, heart and lymphoid organs have cells that express immune-related genes that are different from hemocytes.

Separation of functionally divergent muscle precursor cell populations from porcine juvenile muscles by discontinuous Percoll density gradient centrifugation

BackgroundSatellite cells (SC) and their descendants, muscle precursor cells (MPC), play a key role in postnatal muscle development, regeneration, and plasticity. Several studies have provided evidence that SC and MPC represent a heterogeneous population differing in their biochemical and functional properties. The identification and characterization of functionally divergent SC subpopulations should help to reveal the precise involvement of SC/MPC in these myogenic processes. The aim of the present work was therefore to separate SC subpopulations by using Percoll gradients and to characterize their myogenic marker profiles and their functional properties (adhesion, proliferation, and differentiation).ResultsSC/MPC from muscles of 4-day-old piglets were isolated by trypsin digestion and enriched by Percoll density gradient centrifugation. A mixed myogenic cell population was obtained from the 40/70% interface (termed: mixed P40/70) of a 25/40/70% Percoll gradient. Thereafter, by using a more stepped 25/40/50/70% Percoll gradient, mixed P40/70 was divided into subpopulation 40/50 (SP40/50) collected from the 40/50% interface and subpopulation 50/70 (SP50/70) collected from the 50/70% interface.All three isolated populations proliferated and showed a myogenic phenotype characterized by the ability to express myogenic markers (Pax7, MyoD1, Desmin, and MyoG) and to differentiate into myotubes. However, compared with mixed P40/70, SP40/50 and SP50/70 exhibited distinct functional behavior. Growth kinetic curves over 90 h obtained by the xCELLigence system and proliferation assays revealed that SP40/50 and mixed P40/70 constituted a fast adhering and fast proliferating phenotype. In contrast, SP50/70 showed considerably slower adhesion and proliferation. The fast-proliferating SP40/50 showed the highest myogenic differentiation potential with higher fusion rates and the formation of more middle-sized and large myotubes.ConclusionsThe described Percoll density gradient centrifugation represents a useful tool for subdividing pig SC/MPC populations with divergent myogenic functions. The physiological role of SC subpopulations during myogenesis and the interaction of these populations can now be analyzed to a greater extent, shedding light on postnatal growth variations in pigs and probably in other animals.

Testicular germ line cell identification, isolation, and transplantation in two North American catfish species.

Our aim was to transplant blue catfish germ line stem cells into blastulae of triploid channel catfish embryos to produce interspecific xenogenic catfish. The morphological structure of the gonads of blue catfish (Ictalurus furcatus) in ~ 90- to 100-day-old juveniles, two-year-old juveniles, and mature adults was studied histologically. Both oogonia (12-15μm, diameter with distinct nucleus 7-8μm diameter) and spermatogonia (12-15μm, with distinct nucleus 6-7.5μm diameter) were found in all ages of fish. The percentage of germ line stem cells was higher in younger blue catfish of both sexes. After the testicular tissue was trypsinized, a discontinuous density gradient centrifugation was performed using 70, 45, and 35% Percoll to enrich the percentage of spermatogonial stem cells (SSCs). Four distinct cell bands were generated after the centrifugation. It was estimated that 50% of the total cells in the top band were type A spermatogonia (diameter 12-15μm) and type B spermatogonia (diameter 10-11μm). Germ cells were confirmed with expression of vasa. Blastula-stage embryos of channel catfish (I. punctatus) were injected with freshly dissociated blue catfish testicular germ cells as donor cells for transplantation. Seventeen days after the transplantation, 33.3% of the triploid channel catfish fry were determined to be xenogenic catfish. This transplantation technique was efficient, and these xenogenic channel catfish need to be grown to maturity to verify their reproductive capacity and to verify that for the first time SSCs injected into blastulae were able to migrate to the genital ridge and colonize. These results open the possibility of artificially producing xenogenic channel catfish males that can produce blue catfish sperm and mate with normal channel catfish females naturally. The progeny would be all C × B hybrid catfish, and the efficiency of hybrid catfish production could be improved tremendously in the catfish industry.