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Lipid rafts are usually isolated from cells or tissues using sucrose gradient ultracentrifugation in the presence of detergents such as Triton X-100 at 4 °C. Although detergents should be removed for further structural characterization following fractionation, these compounds are often difficult to completely remove, especially from the glycolipids. In this chapter, we describe a novel method for the fast and convenient removal of detergents from lipid raft glycolipids following fraction and describe the application of this method.

Gram-negative diderm bacteria are characterized by a tripartite cell envelope, composed of an inner membrane (IM) and a lipopolysaccharide (LPS)-containing outer membrane (OM), separated by an aqueous space where the peptidoglycan is embedded. LPS is a peculiar glycolipid endowed with several biological activities. The biosynthesis and transport of LPS to its final location take place in every compartment of the cell envelope. Proteins and protein machineries with different subcellular localization are involved in this process to facilitate the trafficking of LPS across subcellular compartments that differ in their physicochemical proprieties. The fractionation of bacterial cell envelopes can give information on the status of the LPS biogenesis by allowing the analysis of LPS profiles and of the localization of proteins involved in the transport. Here, we describe a standardized protocol for membrane fractionation in Escherichia coli using sucrose density gradient centrifugation that separates the IM from the OM cellular fractions. Bacterial cells are first converted into spheroplasts and lysed; then the membrane fractions are collected by ultracentrifugation and separated at high speed by exploiting the differences in membrane density. The fractions obtained are analyzed for LPS total amount and electrophoretic profile.

Microfluidics and microfilter devices have been developed to mimic the characteristics of the female reproductive tract, minimizing the risk of sperm damage. This study aimed to compare the use of a microfilter device versus conventional methods for sperm selection used in in vitro fertilization (IVF). For selecting spermatozoa, the pooled samples were processed in a microfilter device, swim-up and mini-Percoll gradient. Kinematic and morphometric parameters, vitality and DNA damage were analysed before and after sperm selection. After selection, 10,000 motile spermatozoa per oocyte were used in IVF drops. Embryos were assessed at three (cleavage rate) and seven (blastocyst rate) days post-IVF. Results of sperm kinematic parameters including average path velocity, velocity straight line, curvilinear velocity, linearity, lateral head displacement with the microfilter device were superior to density gradient (p 0.05), but similar to swim-up method. Likewise, sperm DNA damage was significantly reduced using the microfilter device and swim-up method. Regarding the total sperm recovery rate post selection, results with the microfilter device (17.64%) and mini-Percoll gradient (18.27%) were higher than with swim-up method (6.52%). However, the cleavage and blastocyst rates were the lowest using the microfilter device. In conclusion, sperm selection using the microfilter device and swim-up method can improve kinematic parameters, although the mini Percoll gradient was the most efficient method for embryo production.

Raman spectroscopy is an analytical method to identify medical samples of bacteria. Because Raman spectroscopy detects the biochemical properties of a cell, there are many factors that can influence and modify the Raman spectra of bacteria. One possible influence is a proper method for isolation of the bacteria. Medical samples in particular never occur in purified form, so a Raman-compatible isolation method is needed which does not affect the bacteria and thus the resulting spectra. In this study, we present a Raman-compatible method for isolation of bacteria from bronchoalveolar lavage (BAL) fluid using density gradient centrifugation. In addition to measuring the bacteria from a patient sample, the yield and the spectral influence of the isolation on the bacteria were investigated. Bacteria isolated from BAL fluid show additional peaks in comparison to pure culture bacteria, which can be attributed to components in the BAL sample. The isolation gradient itself has no effect on the spectra, and with a yield of 63% and 78%, the method is suitable for isolation of low concentrations of bacteria from a complex matrix. Graphical abstract Supplementary Information The online version contains supplementary material available at 10.1007/s00216-021-03488-0.

Extracellular vesicles (EVs), which are nanosized vesicles released by cells as intracellular messengers, have high potential as biomarkers. EVs are usually collected from in vitro sources, such as cell culture media or biofluids, and not from tissues. Techniques enabling direct collection of EVs from tissues will extend the applications of EVs. We compared methods for separating EVs from solid liver, heart, and skeletal muscle. Compared with a precipitation method, an ultracentrifugation‐based method for collection of EVs from solid tissues yielded a higher proportion of EVs positive for EV‐related markers, with minimum levels of intracellular organelle‐related markers. Some tissue‐specific modifications, such as a sucrose cushion step, may improve the yield and purity of the collected EVs., Extracellular vesicle (EV)‐related research is mostly focused on cell culture and biofluids. Recently published studies reported the collection of EVs from a single tissue type. We compared several separation methods and collected vesicles from three different types of solid tissue—liver, skeletal muscle, and heart. EV characterization followed the criteria of the International Society of Extracellular Vesicles (ISEV).

Extracellular vesicles (EVs) are lipid bilayered membrane structures released by all cells. Most EV studies have been performed by using cell lines or body fluids, but the number of studies on tissue-derived EVs is still limited. Here, we present a protocol to isolate up to six different EV subpopulations directly from tissues. The approach includes enzymatic treatment of dissociated tissues followed by differential ultracentrifugation and density separation. The isolated EV subpopulations are characterized by electron microscopy and RNA profiling. In addition, their protein cargo can be determined with mass spectrometry, western blot and ExoView. Tissue-EV isolation can be performed in 22 h, but a simplified version can be completed in 8 h. Most experiments with the protocol have used human melanoma metastases, but the protocol can be applied to other cancer and non-cancer tissues. The procedure can be adopted by researchers experienced with cell culture and EV isolation.

Sperm preparation is critical to achieving a successful intrauterine insemination and requires the processing of a semen sample to remove white blood cells, wash away seminal plasma, and reduce sample volume. We present an automated instrument capable of performing a sperm preparation starting with a diluted semen sample. We compare our device against a density gradient centrifugation by processing 0.5 mL portions of patient samples through each treatment. In 5 min of operating time, the instrument recovers an average of 86% of all sperm and 82% of progressively motile sperm from the original sample while removing white blood cells, replacing the seminal plasma, and reducing the volume of the sample to the clinically required level. In 25 min of operating time, density gradient centrifugation recovers an average of 33% of all sperm and 41% of progressively motile sperm. The automated instrument could improve access to IUI as a treatment option by allowing satellite doctor’s offices to offer intrauterine insemination as an option for patients without the clinical support required by existing methods.

Background Density gradient centrifugation (DGC) and swim-up (SU) are the two most widely used sperm preparation methods for in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI). However, existing comparisons of IVF/ICSI outcomes following these sperm preparation methods are insufficient and controversial. Methods This retrospective study included all first autologous IVF and ICSI cycles performed between March 1, 2016, and December 31, 2020 in a single university-based center. A total of 3608 cycles were matched between DGC and SU using propensity score (PS) matching for potential confounding factors at a ratio of 1:1. The primary outcome was the cumulative live birth rate (cLBR) per aspiration. Results PS matching provided 719 cycles after DGC and 719 cycles after SU. After adjusting for confounders, the recovery rate, progressive motility rate after sperm preparation, fertilization rate, good-quality embryo rate, and blastocyst formation rate were similar between the DGC and SU groups. The cLBR (odds ratio [OR] = 1.143, 95% confidence interval [CI]: 0.893–1.461) and LBR per transfer (OR = 1.082, 95% CI: 0.896–1.307) were also not significantly different between the groups. Furthermore, no significant differences were found in all of the laboratory and clinical outcomes following conventional IVF or ICSI cycles between the two groups. However, a significantly higher fertilization rate (β = 0.074, 95% CI: 0.008–0.140) was observed when using poor-quality sperm in the DGC group than in the SU group. Conclusions Sperm preparation using DGC and SU separately resulted in similar IVF/ICSI outcomes. Further studies are warranted to compare the effects of these methods on IVF/ICSI outcomes when using sperm from subgroups of different quality.

Fibroblast growth factor receptor (FGFR) is associated with proliferation, migration, and angiogenesis of carcinomas, and FGFR signaling inhibitors are considered a key drug for the treatment of solid tumors with FGFR overexpression. Amplification of FGFR2 is reportedly identified in 3%‐10% of gastric cancers (GCs). The aim of this study is to clarify whether the identification of the circulating tumor cells (CTCs) with FGFR2 overexpression is useful to detect patients with FGFR2‐overexpressing GC. One hundred GC patients who underwent gastrectomy were enrolled. A total volume of 8 mL of peripheral blood was collected from each patient just before gastrectomy, and mononuclear cells were enriched by Ficol density gradient centrifugation. These cells were immunostained with PI/CD45/EpCAM/FGFR2. The number of CTCs with FGFR2 expression in each sample was enumerated by FACScan. The FGFR2 expression level of the resected primary tumor was assessed by immunohistochemistry. The number of FGFR2‐positive CTCs in the GC patients' peripheral blood was significantly correlated with the FGFR2 expression level of the primary GC. The relapse‐free survival of the patients with FGFR2‐positive CTCs (≥5 cells/10 mL blood) was significantly poorer (P = .018, log‐rank) than that of the patients without FGFR2‐positive CTCs (, We aim to clarify the clinical significance of FGFR2‐positive CTCs in gastric cancer. FGFR2‐positive CTCs were correlated with the FGFR2 expression level of primary GC. The detection of FGFR2‐positive CTCs might help identify an existing FGFR2‐positive tumor.

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.

To assist in the study of cellular aging, we established a new method of enriching physiologically aged bone marrow stromal cells (BMSCs) in animals of any age using a Percoll density gradient centrifugation technique. BMSCs from mice 2 months of age were isolated, and their cellular age determined (over 80% Scal-1+ CD29+ CD11b- CD45- CD105- and able to differentiate into osteoblasts, adipocytes, and chondrocytes). As proof –of principle, cells were aged in vitro and confirmed by low bromodeoxyuridine (BrdU) incorporation and senescence-associated β-galactosidase (SA-β-gal) staining. Proliferating cells were enriched in high-density gradient layers, and senescent cells were enriched in low-density gradient layers. We confirmed that over 80% of cells from the low-density gradient layer were senescent with SA-β-gal staining and telomere dysfunction-induced foci (TIF) assay. This density-based method, which can separate proliferating and senescent BMSCs, could be used to study mechanisms of physiologic cell aging and may have implications for the use of BMSCs in clinical transplant applications.

Aim A high-risk strategy has been implemented for lipid-lowering therapy in the primary prevention of cardiovascular disease. However, atherosclerosis and cardiovascular events are common among individuals with low cardiovascular risk. This study aimed to determine whether the small dense low-density lipoprotein cholesterol (sdLDLC) level can predict carotid atherosclerosis progression and identify high-risk individuals. Methods Baseline sdLDLC and low-density lipoprotein cholesterol (LDLC) were measured in 808 participants from the Chinese Multi-provincial Cohort Study, aged 45-74 years. Adjusted relative risk was calculated using a modified Poisson regression model to assess the relationship between sdLDLC and 5-year atherosclerosis progression, as indicated by the progression, incidence, and multi-territorial extent of carotid plaque. Results The 5-year atherosclerosis progression increased significantly with increased sdLDLC. Baseline sdLDLC was significantly associated with the short-term risk of plaque progression after multivariable adjustment, even in participants with low LDLC or a 10-year estimated cardiovascular risk. sdLDLC predicted plaque progression (relative risk 2.05; 95% confidence interval 1.43-2.93) in participants with LDLC ＜130 mg/dL. Furthermore, participants with the highest sdLDLC but intermediate or low cardiovascular risk (accounting for 16% of the cohort) had double the risk of plaque progression, which was comparable to those with the same sdLDLC and high cardiovascular risk, relative to those with the lowest sdLDLC levels and low cardiovascular risk. Conclusions sdLDLC is independently associated with the progression of carotid atherosclerosis, which may provide a basis for clinicians to reclassify individuals believed to be at low cardiovascular risk into the high-risk category, and those with high sdLDLC may benefit from more aggressive cholesterol-lowering treatment.

Subcellular organization of RNAs and proteins is critical for cell function, but we still lack global maps and conceptual frameworks for how these molecules are localized in cells and tissues. Here, we introduce ATLAS-Seq, which generates transcriptomes and proteomes from detergent-free tissue lysates fractionated across a sucrose gradient. Proteomic analysis of fractions confirmed separation of subcellular compartments. Unexpectedly, RNAs tended to co-sediment with other RNAs in similar protein complexes, cellular compartments, or with similar biological functions. With the exception of those encoding secreted proteins, most RNAs sedimented differently than their encoded protein counterparts. To identify RNA binding proteins potentially driving these patterns, we correlated their sedimentation profiles to all RNAs, confirming known interactions and predicting new associations. Hundreds of alternative RNA isoforms exhibited distinct sedimentation patterns across the gradient, despite sharing most of their coding sequence. These observations suggest that transcriptomes can be organized into networks of co-segregating mRNAs encoding functionally related proteins and provide insights into the establishment and maintenance of subcellular organization.

Exosomes have been described as promising biomarkers for understanding disease progression and prognosis. These lipid membrane nanoparticles derived from airway cells have been shown to have immunomodulatory effects, such as driving inflammatory responses in asthma. These emerging evidences demonstrating an important pathophysiological role of exosomes warrants the development of novel approaches for isolation and rapid characterization of exosomes, which would be applicable for both translational and clinical studies. In this review article, we describe two methods of rapid exosomes characterization: (1) imaging flow cytometry using ImageStream; and (2) conventional flow cytometry using the BD Symphony A5 platform. We also explore sorting of exosomes using the BD Aria.

Extracellular vesicles (EVs), derived from cell membranes, demonstrate the potential to be excellent therapeutics and drug carriers. Although EVs are promising, the process to develop high-quality and scalable EVs for their translation is demanding. Within this research, we analyzed the production of EVs, their purification and their post-bioengineering, and we also discussed the biomedical applications of EVs. We focus on the developments of methods in producing EVs including biological, physical, and chemical approaches. Furthermore, we discuss the challenges and the opportunities that arose when we translated EVs in clinic. With the advancements in nanotechnology and immunology, genetically engineering EVs is a new frontier in developing new therapeutics in order to tailor to individuals and different disease stages in treatments of cancer and inflammatory diseases.

Extracellular vesicle (EV) is a unified terminology of membrane-enclosed vesicular species ubiquitously secreted by almost every cell type and present in all body fluids. They carry a cargo of lipids, metabolites, nucleic acids and proteins for their clearance from cells as well as for cell-to-cell communications. The exact composition of EVs and their specific functions are not well understood due to the underdevelopment of the separation protocols, especially those from the central nervous system including animal and human brain tissues as well as cerebrospinal fluids, and the low yield of proteins in the separated EVs. To understand their exact molecular composition and their functional roles, development of the reliable protocols for EV separation is necessary. Here we report the methods for EV separation from human and mouse unfixed frozen brain tissues by a sucrose step gradient ultracentrifugation method, and from human cerebrospinal fluids by an affinity capture method. The separated EVs were assessed for morphological, biophysical and proteomic properties of separated EVs by nanoparticle tracking analysis, transmission electron microscopy, and labeled and label-free mass spectrometry for protein profiling with step-by-step protocols for each assessment.

Background It is not unusual for stallions to have fertility problems. For many, artificial insemination with more dense spermatozoa (isolated by density gradient centrifugation) results in greater pregnancy rates compared with the rates when using unfractionated spermatozoa. RNAs in spermatozoa delivered to the oocyte at conception are required for embryo development. Novel molecular assays of spermatozoa that reflect function are needed to predict the fertility of stallions. Objectives To describe and compare the RNA populations in more dense and less dense spermatozoa from stallions. Materials and methods Spermatozoa from five stallions were separated into more dense and less dense populations by density gradient centrifugation. Complementary DNA libraries were made from each of the ten total RNA samples after ribosomal RNA removal. Next-generation sequencing characterized the RNA populations in more and less dense spermatozoa. Quantitative reverse transcription-PCR was used to confirm differential expression of selected RNAs. Results Stallion spermatozoa contain 11 215 RNAs, with the most prevalent RNA being a 1492 base long non-coding RNA. The levels of 159 RNAs were greater in more dense spermatozoa, while levels of seven other RNAs were greater in less dense spermatozoa. Quantitative reverse transcription-PCR confirmed the threefold greater levels of solute carrier family 26 member 8 (SLC26A8) mRNA in less dense spermatozoa, and sixfold and threefold greater expression levels of the SCP2 sterol binding domain containing 1 (SCP2D1) and spermatogenesis-associated protein 31D1 (SPATA31D1) mRNAs in more dense spermatozoa, respectively. Discussion and conclusion We identified 11 215 RNAs in stallion spermatozoa and 166 with differential expression between more dense and less dense fractions. Many prevalent RNAs were also found in bull, boar, and human spermatozoa. Many differentially expressed RNAs are known to be testis- or spermatozoa-specific. Our results may lead to identification of an RNA population in spermatozoa that is optimal for establishing successful pregnancies.

PURPOSE: To evaluate embryologic outcomes among paired IVF cycles in which a microfluidics chip was utilized compared to density gradient centrifugation for sperm processing. METHODS: This was a retrospective cohort study of 88 paired IVF cycles from patients aged 18–44 years at a university-affiliated IVF center. Fresh cycles from patients undergoing ICSI with sperm processed by a microfluidics chamber (microfluidics cycles) were compared to the same patients’ previous ICSI cycles in which sperm was processed via density gradient centrifugation (control cycles). The primary outcome was the high-quality blastulation rate. RESULTS: High-quality blastulation rate per oocyte retrieved was significantly higher in the microfluidics group compared to the control group (21.1% versus 14.5%, p

(1) Background: When red blood cells are centrifuged in a continuous Percoll-based density gradient, they form discrete bands. While this is a popular approach for red blood cell age separation, the mechanisms involved in banding were unknown. (2) Methods: Percoll centrifugations of red blood cells were performed under various experimental conditions and the resulting distributions analyzed. The age of the red blood cells was measured by determining the protein band 4.1a to 4.1b ratio based on western blots. Red blood cell aggregates, so-called rouleaux, were monitored microscopically. A mathematical model for the centrifugation process was developed. (3) Results: The red blood cell band pattern is reproducible but re-centrifugation of sub-bands reveals a new set of bands. This is caused by red blood cell aggregation. Based on the aggregation, our mathematical model predicts the band formation. Suppression of red blood cell aggregation reduces the band formation. (4) Conclusions: The red blood cell band formation in continuous Percoll density gradients could be explained physically by red blood cell aggregate formation. This aggregate formation distorts the density-based red blood cell age separation. Suppressing aggregation by osmotic swelling has a more severe effect on compromising the RBC age separation to a higher degree.

Background The wisent (Bison bonasus) is a species that has undergone a population bottleneck. Homozygosity is prevalent within the population and may have a negative impact on semen quality in wisent bulls. Semen samples containing a large amount of functionally and morphologically impaired or dead spermatozoa have lower tolerance for cryopreservation process. Such samples are prone to involve damage acrosomes, to produce and release reactive oxygen which negatively affects proper function of spermatozoas. It is a good practice to select intact and viable gametes before subjecting the sample to cryopreservation to improve the efficiency of this process. The aim of this study was to assess the ability of Percoll® density gradient centrifugation in order to improve the quality of wisent spermatozoa after cryopreservation. Spermatozoa samples were analysed with computer-assisted semen analysis system and flow cytometry. Results Percoll® density gradient centrifugation resulted in increased percentage of motile spermatozoa, higher proportion of spermatozoa with normal morphology and proper functionality but also in a significant reduction of the total number of gametes. Nevertheless, the concentration of frozen spermatoza was still sufficient for obtaining a few complete insemination doses suggested for cattle from each epididymis. Conclusions While creating a high-quality genetic reserve, for in vitro fertilisation purposes, eliminating detritus and improving the overall quality of samples is more important than total number of spermatozoa. For these reasons, the achievement of higher post thaw quality of spermatozoa justifies the purification of samples by centrifugation in a Percoll® density gradient prior to the cryopreservation process.

Accurate isolation of functional and intact lysosomes enables the quantification and analyses of abundances, dynamic changes and enrichment levels of lysosomal content, allowing specific lysosomal investigations induced by autophagy. In this protocol chapter, we describe detailed practical instructions and advices for an efficacious lysosomal enrichment and isolation procedure by differential multilayered density gradient centrifugations using human cancer cell lines. By this method, intact and autophagy competent lysosomes can be isolated from cancer cells based on their distinct density and obtained fractions can further be analyzed for functional lysosomal assays, as well as for protein or metabolic loads to identify select spatiotemporal changes by comparative quantitative measurement. This method has been used to enrich lysosomes from a variety of cancer cells with activated chaperone-mediated autophagy, but can be optimized for other cell lines and tissues for multiple autophagy-induced conditions.

Evaluating studies over the past almost 40 years, this review outlines the current knowledge and research gaps in the use of isolated leucocytes in salmonid immunology understanding. This contribution focuses on the techniques used to isolate salmonid immune cells and popular immunological assays. The paper also analyses the use of leucocytes to demonstrate immunomodulation following dietary manipulation, exposure to physical and chemical stressors, effects of pathogens and parasites, vaccine design and application strategies assessment. We also present findings on development of fish immune cell lines and their potential uses in aquaculture immunology. The review recovered 114 studies, where discontinuous density gradient centrifugation (DDGC) with Percoll density gradient was the most popular leucocyte isolation method. Fish head kidney (HK) and peripheral blood (PB) were the main sources of leucocytes, from rainbow trout (Oncorhynchus mykiss) and Atlantic salmon (Salmo salar). Phagocytosis and respiratory burst were the most popular immunological assays. Studies used isolated leucocytes to demonstrate that dietary manipulations enhance fish immunity, while chemical and physical stressors suppress immunity. In addition, parasites, and microbial pathogens depress fish innate immunity and induce pro-inflammatory cytokine gene transcripts production, while vaccines enhance immunity. This review found 10 developed salmonid cell lines, mainly from S. salar and O. mykiss HK tissue, which require fish euthanisation to isolate. In the face of high costs involved with density gradient reagents, the application of hypotonic lysis in conjunction with mico-volume blood methods can potentially reduce research costs, time, and using nonlethal and ethically flexible approaches. Since the targeted literature review for this study retrieved no metabolomics study of leucocytes, indicates that this approach, together with traditional technics and novel flow cytometry could help open new opportunities for in vitro studies in aquaculture immunology and vaccinology.

About 8% of our genome is composed of sequences from Human Endogenous Retroviruses (HERVs). The HERV-K (HML.2) family, here abbreviated HML.2, is able to produce virus particles that were detected in cell lines, malignant tumors and in autoimmune diseases. Parameters and properties of HML.2 released from teratocarcinoma cell lines GH and Tera-1 were investigated in detail. In most experiments, analyzed viruses were purified by density gradient centrifugation. HML.2 structural proteins, reverse transcriptase (RT) activity, viral RNA (vRNA) and particle morphology were analyzed. The HML.2 markers were predominantly detected in fractions with a buoyant density of 1.16 g/cm3. Deglycosylation of TM revealed truncated forms of transmembrane (TM) protein. Free virions and extracellular vesicles (presumably microvesicles—MVs) with HML.2 elements, including budding intermediates, were detected by electron microscopy. Viral elements and assembled virions captured and exported by MVs can boost specific immune responses and trigger immunomodulation in recipient cells. Sequencing of cDNA clones demonstrated exclusive presence of HERV-K108 env in HML.2 from Tera-1 cells. Not counting two recombinant variants, four known env sequences were found in HML.2 from GH cells. Obtained results shed light on parameters and morphology of HML.2. A possible mechanism of HML.2-induced diseases is discussed.

Isolation of purified mitochondria is an essential technique for the analysis of metabolic and cellular functions associated with this vital organelle. Filamentous fungi, such as Neurospora crassa, have been proven to be highly amenable to the analysis of mitochondria, in part due to their rapid growth rate and relative ease of isolation. Here we describe a step-by-step procedure for the isolation of mitochondria from Fusarium species via differential centrifugation and density step-gradient centrifugation, and include methods to overcome potential complications. Mitochondria purified by flotation gradient procedures remain active for functional assays and can be further fractionated for isolation of nucleic acids or ribonucleoprotein particles that retain enzymatic activity.

Objective: Our study aims to retrospectively examine the relationship between two different sperm preparation methods used in IUI among eight years in terms of pregnancy and live birth rates. Methods: We evaluated the data of semen samples between December 2012 and March 2020. Three hundred eighty-four samples prepared with Conventional Swim-up (CSW) and 361 samples prepared with Density Gradient-Swim up (DGC-SW) obtained from men applying for IUI were analyzed. Spermiogram results of the semen samples given by men applying for IUI were examined. Data about sperm preparation method, post washed sperm parameters, pregnancy, and live birth rate were collected. Statistical analysis was performed. Results: Basal progressive sperm count was significantly higher in pregnant couples in both CSW and DGCSW groups (p = 0,032, p = 0,035, respectively). In each group, the post washed total progressive motile sperm count obtained by CSW and DGC-SW methods were significantly higher in pregnant patients (p < 0.05). There was no significant difference between CSW and DGC-SW methods in pregnancy achievement (p = 0,399, X-2 = 0,712). Live birth and miscarriage rates were not different between the groups (p = 0,243, X-2 = 2.827). Conclusion: In conclusion, there is no significant difference between CSW and DGC-SW for pregnancy and live birth rates. Our results suggest that both sperm preparation techniques used in IUI are not superior to each other. In other words, the choice of sperm preparation method does not affect the pregnancy rate in couples undergoing IUI.(c) 2022 Elsevier Masson SAS. All rights reserved.

Summary This protocol describes how to identify Dual Expressers (DEs), a rare type of lymphocyte that co-expresses B-cell receptors and T-cell receptors, by flow cytometry using a cocktail of four antibodies. It also shows the subsequent gating strategy for detecting and sorting DEs and the generation of EBV-immortalized DE lymphoblastoid cell lines and clones for antibody production and cloning antigen receptors. Use of this protocol maximizes detection of DEs and minimizes inclusion of doublets. For complete details on the use and execution of this protocol, please refer to Ahmed et al. (2019)., Graphical abstract, Highlights • Protocol to detect DEs and distinguish from T and B cells by flow cytometry • Sequential gating strategy to maximize and optimize detection of DEs • Strategy for efficient FACS-sorting and checking purity of sorted DEs • Protocol for generating EBV-immortalized DE lines and clones, This protocol describes how to identify Dual Expressers, a rare type of lymphocyte that co-expresses B-cell receptors and T-cell receptors, by flow cytometry using a cocktail of four antibodies. It also shows the subsequent gating strategy for detecting and sorting DEs and the generation of EBV-immortalized DE lymphoblastoid cell lines and clones for antibody production and cloning antigen receptors. Use of this protocol maximizes detection of DEs and minimizes inclusion of doublets.

The isolation of mitochondria from potato tubers (Solanum tuberosum L.) is described, but the methodology can easily be adapted to other storage tissues. After homogenization of the tissue, filtration and differential centrifugation, the key step is a Percoll density gradient centrifugation. The Percoll gradient contains two parts: a bottom part containing Percoll in 0.3 M sucrose, and a slightly less dense top part containing Percoll in 0.3 M mannitol. After centrifugation, a density gradient is formed that is almost linear in the central part, and this is where the band containing the purified intact mitochondria is formed. This method makes it possible to process large amounts of plant material (2-6 kg) and saves at least 1.5 h on the preparation time compared to methods where two consecutive purification methods are used. Nonetheless, it yields large amounts of mitochondria (50-125 mg protein) of very high purity, intactness and functionality.

The study of plant mitochondria often requires isolation of mitochondria from plant tissues in intact and functional form. Here, we describe an effective procedure of mitochondrial isolation from leaf tissues and whole seedlings of the model dicot species Arabidopsis thaliana by using differential centrifugation and continuous Percoll density gradients.

Background Neutrophils are important for immune surveillance of tumour cells. Neutrophils may also be epigenetically programmed in the tumour microenvironment to promote tumour progression. In addition to the commonly known high-density neutrophils (HDN) based on their separation on density gradient, recent studies have reported the presence of high levels of low-density neutrophils (LDN) in tumour-bearing mice and cancer patients. We reported previously that estrogen promotes the growth of estrogen receptor α-negative mammary tumours in mice undergoing mammary involution through stimulating pro-tumoral activities of neutrophils in the mammary tissue. Methods Female BALB/cAnNTac mice at 7–8 weeks old were mated and bilateral ovariectomy was performed 2 days post-partum. At 24 h after forced-weaning of pups to induce mammary involution, post-partum female mice were injected with either E2V, or vehicle control on alternative days for 2-weeks. On 48 h post-weaning, treated female mice were inoculated subcutaneously with 4 T1-Luc2 cells into the 9th abdominal mammary gland. Age-matched nulliparous female was treated similarly. Animals were euthanized on day 14 post-tumour inoculation for analysis. To evaluate the short-term effect of estrogen, post-partum females were treated with only one dose of E2V on day 12 post-tumour inoculation. Results Estrogen treatment for 2-weeks reduces the number of blood LDN by more than 10-fold in tumour-bearing nulliparous and involuting mice, whilst it had no significant effect on blood HDN. The effect on tumour-bearing mice is associated with reduced number of mitotic neutrophils in the bone marrow and increased apoptosis in blood neutrophils. Since estrogen enhanced tumour growth in involuting mice, but not in nulliparous mice, we assessed the effect of estrogen on the gene expression associated with pro-tumoral activities of neutrophils. Whilst 48 h treatment with estrogen had no effect, 2-weeks treatment significantly increased the expression of Arg1, Il1b and Tgfb1 in both HDN and LDN of involuting mice. In contrast, estrogen increased the expression of Arg1 and Ccl5 in HDN and LDN of nulliparous mice. Conclusions Prolonged estrogenic stimulation in tumour-bearing mice markedly hampered tumour-associated increase of LDN plausibly by inhibiting their output from the bone marrow and by shortening their life span. Estrogen also alters the gene expression in neutrophils that is not seen in tumour-free mice. The results imply that estrogen may significantly influence the tumour-modulating activity of blood neutrophils.

During the in vitro fertilization (IVF) process, sperm must be processed prior to insemination. While the most common method, density gradient centrifugation, can potentially damage sperm during centrifugation, a recent advancement in sperm processing uses a microfluidics system which selects for the most highly motile sperm. In selecting for these sperm which may be of higher quality, the euploidy rates of embryos created as a result may also be improved. The primary aim of this study is to compare the euploidy rates per mature oocyte between embryos created from sibling oocytes injected with sperm processed by microfluidics sorting or by density gradient centrifugation.This is a double-blinded prospective randomized sibling oocyte study including patients undergoing treatment with IVF with intracytoplasmic sperm injection (ICSI) and preimplantation genetic testing (PGT). After controlled ovarian hyperstimulation, oocytes from each patient will be separated into two groups. Each group will be randomized to sperm processed using either microfluidics or density gradient centrifugation and embryos biopsied for PGT to assess euploidy rates. A sample size of 686 oocytes in each group for a total of 1372 oocytes will provide 80% power to detect a significant difference in the euploidy rates per mature oocyte between the two groups. An ancillary study examining the relationship between sperm processing method and sperm DNA fragmentation will be assessed.This study will offer insight into the sperm's contribution to embryo euploidy, and has the potential to provide an alternative method of improving euploidy rates in clinical practice.

This protocol describes a method to obtain subcellular protein fractions from mammalian cells using a combination of detergents, mechanical lysis, and isopycnic density gradient centrifugation. The major advantage of this procedure is that it does not rely on the sole use of solubilizing detergents to obtain subcellular fractions. This makes it possible to separate the plasma membrane from other membrane-bound organelles of the cell. This procedure will facilitate the determination of protein localization in cells with a reproducible, scalable, and selective method. This method has been successfully used to isolate cytosolic, nuclear, mitochondrial, and plasma membrane proteins from the human monocyte cell line, U937. Although optimized for this cell line, this procedure may serve as a suitable starting point for the subcellular fractionation of other cell lines. Potential pitfalls of the procedure and how to avoid them are discussed as are alterations that may need to be considered for other cell lines.

Separating extracellular vesicles (EV) from blood plasma is challenging and complicates their biological understanding and biomarker development. In this study, we fractionate blood plasma by combining size‐exclusion chromatography (SEC) and OptiPrep density gradient centrifugation to study clinical context‐dependent and time‐dependent variations in the biomolecular landscape of systemically circulating EV. Using pooled blood plasma samples from breast cancer patients, we first demonstrate the technical repeatability of blood plasma fractionation. Using serial blood plasma samples from HIV and ovarian cancer patients (n = 10) we next show that EV carry a clinical context‐dependent and/or time‐dependent protein and small RNA composition, including miRNA and tRNA. In addition, differential analysis of blood plasma fractions provides a catalogue of putative proteins not associated with systemically circulating EV. In conclusion, the implementation of blood plasma fractionation allows to advance the biological understanding and biomarker development of systemically circulating EV.

Elucidating the mechanisms contributing to the dysregulated host response to infection as part of the syndrome is a current challenge in sepsis research. Peripheral blood mononuclear cells are widely used in immunological studies. Density gradient centrifugation, a common method, is of limited use for blood drawn from patients with sepsis. A significant number of low-density granulocytes co-purify contributing to low purity of isolated peripheral blood mononuclear cells. Whole blood anticoagulated with lithium heparin was drawn from patients with sepsis (n=14) and healthy volunteers (n=11). Immediately after drawing, the plasma fraction was removed and PBMC were isolated from the cellular fraction by density gradient centrifugation. Samples derived from patients with sepsis were subsequently incubated with cluster of differentiation 15 MicroBeads and granulocytes were depleted using magnetic-activated cell sorting. Core cellular functions as antigen presentation and cytokine secretion were analyzed in cells isolated from healthy volunteers (n=3) before and after depletion to confirm consistent functionality. We report here that depleting CD15+ cells after density gradient centrifugation is a feasible way to get rid of the low-density granulocyte contamination. Afterwards, the purity of isolated, functionally intact peripheral blood mononuclear cells is comparable to healthy volunteers. Information on the isolation purity and identification of the containing cell types are necessary for good comparability between different studies. Depletion of CD15+ cells after density gradient centrifugation is an easy but highly efficient way to gain a higher quality and more reliability in studies using peripheral blood mononuclear cells from septic patients without affecting the functionality of the cells.

Extracellular vesicles (EVs) are increasingly tested as therapeutic vehicles and biomarkers, but still EV subtypes are not fully characterised. To isolate EVs with few co‐isolated entities, a combination of methods is needed. However, this is time‐consuming and requires large sample volumes, often not feasible in most clinical studies or in studies where small sample volumes are available. Therefore, we compared EVs rendered by five commonly used methods based on different principles from conditioned cell medium and 250 μl or 3 ml plasma, that is, precipitation (ExoQuick ULTRA), membrane affinity (exoEasy Maxi Kit), size‐exclusion chromatography (qEVoriginal), iodixanol gradient (OptiPrep), and phosphatidylserine affinity (MagCapture). EVs were characterised by electron microscopy, Nanoparticle Tracking Analysis, Bioanalyzer, flow cytometry, and LC‐MS/MS. The different methods yielded samples of different morphology, particle size, and proteomic profile. For the conditioned medium, Izon 35 isolated the highest number of EV proteins followed by exoEasy, which also isolated fewer non‐EV proteins. For the plasma samples, exoEasy isolated a high number of EV proteins and few non‐EV proteins, while Izon 70 isolated the most EV proteins. We conclude that no method is perfect for all studies, rather, different methods are suited depending on sample type and interest in EV subtype, in addition to sample volume and budget.

Stocks of vesicular stomatitis virus free of defective interfering particles were produced by serial clonal isolation. High-multiplicity infections with these stocks led to no interference or formation of defective interfering particles. Defective interfering particles were generated by three successive passages at high multiplicity.

Polysome fractionation by sucrose density gradient centrifugation is a powerful tool that can be used to create ribosome profiles, identify specific mRNAs being translated by ribosomes, and analyze polysome associated factors. While automated gradient makers and gradient fractionation systems are commonly used with this technique, these systems are generally expensive and can be cost-prohibitive for laboratories which have limited resources or cannot justify the expense due to their infrequent or occasional need to perform this method for their research. Here a protocol is presented to reproducibly generate polysome profiles using standard equipment available in most molecular biology laboratories without the use of specialized fractionation instruments. Moreover, a comparison of polysome profiles generated with and without a gradient fractionation system is provided and strategies to optimize and produce reproducible polysome profiles are discussed. Saccharomyces cerevisiae is utilized as a model organism in this protocol, but this protocol can be easily modified and adapted to generate ribosome profiles for many different organisms and cell types.