Iodixanol Density Gradient Centrifugation Research Articles

Suspension Culture Production and Purification of Adeno-Associated Virus by Iodixanol Density Gradient Centrifugation for In Vivo Applications.

This protocol describes recombinant adeno-associated virus (rAAV) production and purification by iodixanol density gradient centrifugation, a serotype-agnostic method of purifying AAV first described in 1999. rAAV vectors are widely used in gene therapy applications to deliver transgenes to various human cell types. In this work, the recombinant virus is produced by transfection of Expi293 cells in suspension culture with plasmids encoding the transgene, vector capsid, and adenoviral helper genes. Iodixanol density gradient centrifugation purifies full AAV particles based on particle density. Additionally, three steps are included in this now-ubiquitous methodology in order to increase total virus yield, decrease the risk of precipitation due to contaminating proteins, and further concentrate the final virus product, respectively: precipitation of viral particles from cell media using a solution of polyethylene glycol (PEG) and sodium chloride, the introduction of a second round of iodixanol density gradient centrifugation, and buffer exchange via a centrifugal filter. Using this method, it is possible to consistently achieve titers in the range of 1012 viral particles/mL of exceptional purity for in vivo use.

Low-density small extracellular vesicles in bovine follicular fluid carrying let-7i target FASLG to inhibit granulosa cells apoptosis

Apoptosis of granulosa cells is a key factor in mammalian follicular atresia. It has a significant impact on oocyte development and maturation. Extracellular vesicles (EVs) are a group of highly heterogeneous population. Previous studies have found that ovarian follicular fluid is rich in EVs. In the present study, the follicular fluid of 3–5 mm follicles from bovine ovaries without corpus luteum was collected, and a subtype of small EVs (sEVs), low-density small EVs (LD-sEVs), was successfully isolated by differential ultracentrifugation combined with iodixanol density gradient centrifugation. LD-sEVs were identified using transmission electron microscope, nanoparticle tracking analysis and Western blot. Flow cytometry, Quantitative reverse transcription PCR (RT-qPCR), Western blot, and other methods were used to detect the effect of LD-sEVs on follicular granulosa cell apoptosis. After that, let-7i, a highly expressed miRNA component in LD-sEVs, was screened and target validation was carried out in granulosa cells. The results showed that LD-sEVs could be taken up by granulosa cells and inhibited the apoptosis. Further research found that let-7i inhibits the apoptosis of granulosa cells by targeting FASLG. It plays an important role in regulating the apoptosis of follicular granulosa cells, which may affect follicular development.

Investigation of canine extracellular vesicles in diffuse large B-cell lymphomas.

Diffuse large B-cell lymphomas (DLBCLs) are the most common lymphoproliferative diseases in dogs. DLBCL diagnosis to date has relied on histopathological analysis; however liquid biopsies have gained attention in recent years as a source of diagnostic and prognostic information. Liquid biopsies can be a source of circulating DNA, miRNA, circulating tumour cells or extracellular vesicles (EVs). In this study EVs were isolated from the plasma of healthy dogs, and dogs with lymphoma, and adenocarcinoma by iodixanol density gradient centrifugation. These EVs were positive for the EV markers CD63 and TSG101 and the pan-B cell markers CD79a, CD21, CD45, CD20. NTA analysis revealed that the DLBCL and adenocarcinoma dogs had elevated plasma EVs relative to the healthy dogs. Furthermore, the modal size of lymphoma EVs had decreased relative to healthy dogs while adenocarcinoma EVs were unchanged. This study demonstrates that the plasma EV population is altered in canine lymphoma patients in a manner similar to previous studies on human lymphomas. The similar changes to the EV population in dogs, together with the similar pathological features and treatment protocols in canine and human non-Hodgkin lymphomas would make dogs a good comparative model for studying the role of EVs in DLBCL development and progression.

Iodixanol density gradients as an effective phytoplasma enrichment approach to improve genome sequencing

Obtaining complete phytoplasma genomes is difficult due to the lack of a culture system for these bacteria. To improve genome assembly, a non-ionic, low- and iso-osmotic iodixanol (Optiprep™) density gradient centrifugation method was developed to enrich for phytoplasma cells and deplete plant host tissues prior to deoxyribonucleic acid (DNA) extraction and high-throughput sequencing (HTS). After density gradient enrichment, potato infected with a ‘Candidatus Phytoplasma australasia’-related strain showed a ∼14-fold increase in phytoplasma HTS reads, with a ∼1.7-fold decrease in host genomic reads compared to the DNA extracted from the same sample without density gradient centrifugation enrichment. Additionally, phytoplasma genome assemblies from libraries equalized to 5 million reads were, on average, ∼15,000 bp larger and more contiguous (N50 ∼14,800 bp larger) than assemblies from the DNA extracted from the infected potato without enrichment. The method was repeated on capsicum infected with Sweet Potato Little Leaf phytoplasma (‘Ca. Phytoplasma australasia’-related strain) with a lower phytoplasma titer than the potato. In capsicum, ∼threefold more phytoplasma reads and ∼twofold less host genomic reads were obtained, with the genome assembly size and N50 values from libraries equalized to 3.4 million reads ∼137,000 and ∼4,000 bp larger, respectively, compared to the DNA extracted from infected capsicum without enrichment. Phytoplasmas from potato and capsicum were both enriched at a density of 1.049–1.058 g/ml. Finally, we present two highly contiguous ‘Ca. Phytoplasma australasia’ phytoplasma reference genomes sequenced from naturally infected Solanaceae hosts in Australia. Obtaining high-quality phytoplasma genomes from naturally infected hosts will improve insights into phytoplasma taxonomy, which will improve their detection and disease management.

Isolation of circulating exosomes and identification of exosomal PD-L1 for predicting immunotherapy response.

Exosomes, a subgroup of extracellular vesicles secreted by multiple cells, have great potential as cancer biomarkers in clinical applications. However, enrichment and detection of exosomes from complex media remain a huge challenge due to their small size. Herein, we used iodixanol density gradient centrifugation for the isolation and purification of exosomes and label-free detection of exosomal PD-L1 using a biochip based on surface plasmon resonance (SPR-ExoPD-L1). The obtained exosomes are lipid-bilayer vesicles and the classical exosome markers CD9, CD63 and CD81 are highly enriched. Besides, PD-L1 is specifically expressed on exosomes instead of non-vesicular components or large extracellular vesicles. Compared with enzyme-linked immunosorbent assays, the SPR-ExoPD-L1 assay could better distinguish exosomes derived from melanoma cells with different levels of PD-L1. Accurate measurement of exosomal PD-L1 could provide critical clinical information for cancer diagnosis and personalized immunotherapy of cancer.

Unraveling mechanisms of pentraxin 3 secretion in adipocytes during inflammation.

Pentraxin 3 (PTX3) is a soluble pattern recognition receptor playing an important role in immune response and inflammation. Lipopolysaccharide (LPS) stimulation can significantly induce PTX3 expression and secretion in adipocytes. Appropriate regulation of PTX3 secretion is critical for inflammatory homeostasis. Using chemical inhibitors of conventional and unconventional protein secretion, we explored the mechanisms that control LPS-stimulated PTX3 secretion in 3T3-L1 adipocytes. Inhibiting the conventional protein secretion blocked LPS-stimulated PTX3 secretion, resulting in cellular PTX3 accumulation in adipocytes. We also detected PTX3 in exosomes from LPS-treated adipocytes; inhibiting exosome trafficking attenuated PTX3 secretion. However, only 4.3% of secreted PTX3 was detected in exosomes compared to 95.7% in the non-exosomal fractions. The fractionation of isolated exosomes by the iodixanol density gradient centrifugation confirmed that a small portion of secreted PTX3 overlapped with exosomal markers in small extracellular-vesicle fractions. We conclude that PTX3 is secreted mainly through conventional protein secretion, and a small percentage of PTX3 is released in exosomes from LPS-stimulated adipocytes.

Colchicine reduces extracellular vesicle NLRP3 inflammasome protein levels in chronic coronary disease: A LoDoCo2 biomarker substudy

Background and aimsColchicine reduces the risk of cardiovascular events in patients with coronary disease. Colchicine has broad anti-inflammatory effects and part of the atheroprotective effects have been suggested to be the result of NLRP3 inflammasome inhibition. We studied the effect of colchicine on extracellular vesicle (EV) NLRP3 protein levels and inflammatory markers, high sensitivity-CRP (hs-CRP) and interleukin (IL)-6, in patients with chronic coronary disease. MethodsIn vitro, the NLRP3 inflammasome was stimulated in PMA-differentiated- and undifferentiated THP-1 cells. In vivo, measurements were performed in serum obtained from 278 participants of the LoDoCo2 trial, one year after randomization to colchicine 0.5 mg once daily or placebo. EVs were isolated using precipitation. NLRP3 protein presence in EVs was confirmed using iodixanol density gradient centrifugation. Levels of NLRP3 protein, hs-CRP and IL-6 were measured using ELISA. ResultsIn vitro, NLRP3 inflammasome stimulation showed an increase of EV NLRP3 protein levels.EV NLRP3 protein levels were lower in patients treated with colchicine (median 1.38 ng/mL), compared to placebo (median 1.58 ng/mL) (p = 0.025). No difference was observed in serum NLRP3 protein levels. Serum hs-CRP levels were lower in patients treated with colchicine (median 0.80 mg/L) compared to placebo (median 1.34 mg/L) (p < 0.005). IL-6 levels were lower in patients treated with colchicine (median 2.07 ng/L) compared to placebo (median 2.59 ng/L), although this was not statistically significant (p = 0.076). ConclusionsColchicine leads to a reduction of EV NLRP3 protein levels. This indicates that inhibitory effects on the NLRP3 inflammasome might contribute to the atheroprotective effects of colchicine in coronary disease.

Unraveling Secretory Mechanisms that Control Pentraxin 3 Secretion in Adipocytes During Inflammation

As a soluble pattern recognition receptor, Pentraxin 3 (PTX3) plays an important role in innate immunity and obesity-associated metabolic inflammation. PTX3 is abundantly expressed and secreted in adipocytes in response to lipopolysaccharide (LPS) stimulation. Appropriate regulation of PTX3 secretion is critical for maintaining inflammatory homeostasis. This study aims to unravel the mechanisms that control PTX3 secretion in adipocytes during LPS-induced inflammation. Upon 6h treatment of LPS, PTX3 expression and secretion were significantly induced in 3T3-L1 and stromal-vascular (SV) differentiated adipocytes, but to a lesser extent in SV cells or 3T3-L1 fibroblasts. However, LPSdoes not significantly stimulate PTX3 expression and secretion in macrophages. Using chemical inhibitors of conventional and unconventional protein secretion, we explored the mechanisms for controlling LPS-stimulated PTX3 secretion. 3T3-L1 adipocytes were treated with LPS for 6h in the presence or absence of various inhibitors blocking protein secretion from the Golgi complex (Monensin and Brefeldin A), mitochondrial oxidation (carbonyl cyanide 3-chlorophenylhydrazone [CCCP]), autophagy-lysosome (chloroquine and 3-methyladenine) and inflammasome (Bay 11–7082 and wedelolactone) activation, or exosome synthesis and trafficking (GW4869, manumycin A, calpeptin, and Y-27632). There were no significant effects of all inhibitors except for Monensin, Brefeldin A, and CCCP on intracellular and secreted levels of PTX3 in adipocytes. We found that Monensin and Brefeldin A significantly blocked LPS-stimulated PTX3 secretion, resulting in cellular PTX3 accumulation in adipocytes. Disrupting mitochondrial membrane potential by CCCP caused the reduction in PTX3 secretion from adipocytes. Additionally, we detected PTX3 in exosomes isolated from LPS-treated adipocytes. Inhibiting exosome synthesis by Manumycin A attenuated LPS-stimulated PTX3 secretion in both adipocyte culture media and isolated exosomes but not in the non-exosomal fraction of media, suggesting the involvement of the exosomal pathway in PTX3 secretion. However, the levels of exosomal PTX3 were significantly lower than that of the non-exosomal PTX3, and only 4.3% of secreted PTX3 was detected in the exosomal fraction of cultural media. Inhibiting the Golgi complex pathway blocked both the exosomal and non-exosomal secretion of PTX3 in adipocytes. After further fractionation of isolated crude exosomes by the iodixanol density gradient centrifugation, we showed that the majority of PTX3 was found in the non-extracellular vesicular (EV) fractions; only a small portion of secreted PTX3 overlapped with the exosomal marker CD63 in the small EV fractions. We conclude that PTX3 is secreted mainly through the conventional protein secretion pathway and minimally through the exosomal or EV pathway in response to LPS stimulation.

Isolation of Nuclei from Mouse Dorsal Root Ganglia for Single-nucleus Genomics.

Primary somatosensory neurons, whose cell bodies reside in the dorsal root ganglion (DRG) and trigeminal ganglion, are specialized to transmit sensory information from the periphery to the central nervous system. Our molecular understanding of peripheral sensory neurons has been limited by both their heterogeneity and low abundance compared with non-neuronal cell types in sensory ganglia. We describe a protocol to isolate nuclei from mouse DRGs using iodixanol density gradient centrifugation, which enriches for neuronal nuclei while still sampling non-neuronal cells such as satellite glia and Schwann cells. This protocol is compatible with a range of downstream applications such as single-nucleus transcriptional and epigenomic assays.

Hepatitis E virus replication in human intestinal cells

ObjectiveHepatitis E virus (HEV), one of the most common agent of acute hepatitis worldwide, is mainly transmitted enterically, via contaminated water for HEV genotypes 1 (HEV1) and HEV2, or by...

Helper-free Production of Laboratory Grade AAV and Purification by Iodixanol Density Gradient Centrifugation

Adeno-associated virus (AAV) is one of the most promising gene therapy vectors and is widely used as a gene delivery vehicle for basic research. As AAV continues to become the vector of choice, it is increasingly important for new researchers to have access to a simplified production and purification protocol for laboratory grade recombinant AAV. Here we report a detailed protocol for serotype independent production of AAV using a helper-free HEK293 cell system followed by iodixanol gradient purification, a method described earlier.1 While the core principals of this mammalian AAV production system are unchanged, there have been significant advancements in the production and purification procedure that serve to boost yield, maximize efficiency, and increase the purity of AAV preps. Using this protocol, we are able to constantly obtain high quantities of laboratory grade AAV particles (>5 × 1012 vg) in a week’s time, largely independent of serotype.

Measurement of Cholesterol Transfer from Lysosome to Peroxisome Using an In Vitro Reconstitution Assay.

Low-density lipoproteins (LDLs) are taken up by the cell mainly through receptor-mediated endocytosis. LDL-derived cholesterol leaves lysosome and further transports to downstream organelles for specific cellular needs. We recently report that cholesterol transfers from lysosome to peroxisome through lysosome-peroxisome membrane contact (LPMC). Here, we use iodixanol density gradient centrifugation to isolate lysosomes and peroxisomes separately for the in vitro reconstitution of LPMC. We also apply 3H-cholesterol-labeled lysosomes and peroxisomes in vitro to measure 3H-cholesterol transfer through LPMC.

Production and Characterization of Vectors Based on the Cardiotropic AAV Serotype 9.

Vectors based on adeno-associated virus serotype 9 (AAV9) efficiently transduce cardiomyocytes in both rodents and large animal models upon either systemic or regional vector delivery. In this chapter, we describe the most widely used production and purification method of AAV9. This production approach does not depend on the use of a helpervirus but instead on transient transfection of HEK293T cells with a plasmid containing the recombinant AAV genome and a second plasmid encoding the AAV9 capsid proteins, the AAV Rep proteins and the adenoviral helper functions. The recombinant AAV is then purified by iodixanol density gradient centrifugation. This chapter also describes in detail the characterization and quality control methods required for assuring high quality vector preparations, which is of particular importance for experiments in large animal models.

Kinetic analysis of internalization of white spot syndrome virus by haemocyte subpopulations of penaeid shrimp, Litopenaeus vannamei (Boone), and the outcome for virus and cell.

Little is known about the innate antiviral defence of shrimp haemocytes. In this context, the haemocytes of penaeid shrimp Litopenaeus vannamei (Boone) were separated by iodixanol density gradient centrifugation into five subpopulations (sub): sub 1 (hyalinocytes), sub 2 and 3 (prohyalinocytes), sub 4 (semigranulocytes) and sub 5 (granulocytes) and exposed to beads, white spot syndrome virus (WSSV) and ultraviolet (UV)-killed WSSV. In a first experiment, the uptake of beads, white spot syndrome virus (WSSV) and UV-killed WSSV by these different haemocyte subpopulations was investigated using confocal microscopy. Only haemocytes of sub 1, 4 and 5 were internalizing beads, WSSV and UV-killed WSSV. Beads were engulfed by a much larger percentage of cells (91.2 in sub 1; 84.1 in sub 4 and 58.1 in sub 5) compared to WSSV (9.6 in sub 1; 10.5 in sub 4 and 7.9 in sub 5) and UV-killed WSSV (12.9 in sub 1; 13.3 in sub 4; and 11.8 in sub 5). In a second experiment, it was shown that upon internalization, WSS virions lost their envelope most probably by fusion with the cellular membrane of the endosome (starting between 30 and 60min post-inoculation) and that afterwards the capsid started to become disintegrated (from 360min post-inoculation). Expression of new viral proteins was not observed. Incubation of haemocyte subpopulations with WSSV but not with UV-killed WSSV and polystyrene beads resulted in a significant drop in haemocyte viability. To find the underlying mechanism, a third experiment was performed in which haemocyte subpopulations were exposed to a short WSSV DNA fragment (VP19) and CpG ODNs. These small DNA fragments induced cell death. In conclusion, WSSV is efficiently internalized by hyalinocytes, semigranulocytes and granulocytes, after which the virus loses its envelope; as soon as the capsids start to disintegrate, cell death is activated, which in part may be explained by the exposure of viral DNA to cellular-sensing molecules.

Sperm cleanup and centrifugation processing for cryopreservation.

Fertility rates with artificial insemination are highest with good-quality sperm samples. Therefore, nonviable sperm, cellular debris, and seminal plasma are preferably removed from semen samples prior to use or for preservation. Such compounds are sources where reactive oxygen species are generated during storage or upon cryopreservation, impairing sperm function. In this chapter we describe methods to remove seminal plasma and cellular debris from sperm samples, and for selecting morphologically normal motile sperm. The methods that are described here include: ordinary centrifugation, sperm swim-up, glass wool and Sephadex filtration/adherence, and single-layer as well as discontinuous two-layer iodixanol density gradient centrifugation.

The experiment study of islet isolation and purification from mice pancreas

Objective To investigate the stable and efficient method of islet isolation and purification from mouse pancreas.Methods Collagenase is injected into the common bile duct (means perfusion method) and the pancreatic pieces are enzymatically digested in a collagenase solution (means pieces method).Mouse islets were isolated by the two approaches of collagenase digestion and purificated by iodixanol and Ficoll density gradient centrifugation.The production and purity of the islets were checked by Dithizone immunofluorescence staining.The vitality of the islets was detected by inducing insulin secretion through glucose.Results Comparing the two approaches collagenase digestion,islet yield was significantly higher in the experiments of perfusion method,298.00 ± 15.58 versus 36.00 ± 3.89 islets (P ＜ 0.01).Comparing the Ficoll density gradient centrifugation,iodixanol density gradient centrifugation provided with purity ＞ 90％ (P ＞ 0.05) and greater vitality of islets,the insulin release (3.120 ± 0.046) versus (3.060 ± 0.426) μg/L (P ＞0.05) with 2.75 mmol/L concentration glucose and (11.36 ±0.82) versus (7.98 ±0.56) μg/L (P ＜0.05) with 16.50 mmol/L concentration glucose.Conclusion Collagenase perfusion method provided a greater number of islets and iodixanol purification obtained greater vitality of islets.Collagenase perfusion and iodixanol density gradient are desired method to a desired outcome of islet isolation in both quantity and quality from mice pancreas. Key words: Islet; Isolation; Purification

Separation of Penaeus vannamei haemocyte subpopulations by iodixanol density gradient centrifugation

Methodologies for separation of immune cell subpopulations are essential tools in immunology studies. Up to date, only one methodology for separating crustacean haemocyte subpopulations using Percoll density gradient centrifugation has been described.In the present work, a new methodology to separate Penaeus vannamei haemocyte subpopulations was developed, using a two-step iodixanol density gradient centrifugation. P. vannamei haemolymph was collected with anticoagulant and centrifuged through a first gradient (densities from 1.063 to 1.109g/ml) for 10min at 2000g. Three bands were formed: two bands with lower density close together, and a third band with higher density. The first two were collected together whilst the third band was collected separately. The volume fraction in-between these bands contained dispersed cells and was also collected. The suspension containing the mixture of the first two bands was centrifuged through a second gradient (densities from 1.047 to1.087g/ml) for 15min at 2000g. Two bands were formed and collected individually. All the cell suspensions were used for in vitro culture (cell survival evaluation) and for evaluation of cell morphology by flow cytometry and light microscopy. Each of the three bands contained a major cell type with distinct morphology and behaviour. The dispersed cell fraction contained a mixture of two different cell types, which were distinct from the cell types in the bands. By order of appearance from the top of the gradient, the cell types were named: subpopulations (Sub) 1 (band 1), Sub 2 (band 2), Sub 3+4 (dispersed cells) and Sub 5 (band 3). The purity level (percentage of the major cell type) of Sub 1, 2 and 5 was 95.0±1.0%, 97.7±1.2% and 99.4±0.8%, respectively. Cells of Sub 2 showed the best survival time in vitro (up to 96h) followed by cells from Sub 1, Sub 3+4 and Sub 5. Phagocytic activity was detected in Sub 1 and 4.This methodology allowed the separation and characterization of five morphologically distinct and physiologically active P. vannamei haemocyte subpopulations, from which three were isolated with a very high degree of purity. Therefore, we consider this methodology a valuable alternative for the traditional crustacean haemocyte separation procedure in Percoll.

Purification of white spot syndrome virus by iodixanol density gradient centrifugation

Up to now, only a few brief procedures for purifying white spot syndrome virus (WSSV) have been described. They were mainly based on sucrose, NaBr and CsCl density gradient centrifugation. This work describes for the first time the purification of WSSV through iodixanol density gradients, using virus isolated from infected tissues and haemolymph of Penaeus vannamei (Boone). The purification from tissues included a concentration step by centrifugation (2.5 h at 60,000 g) onto a 50% iodixanol cushion and a purification step by centrifugation (3 h at 80,000 g) through a discontinuous iodixanol gradient (phosphate-buffered saline, 5%, 10%, 15% and 20%). The purification from infected haemolymph enclosed a dialysis step with a membrane of 1,000 kDa (18 h) and a purification step through the earlier iodixanol gradient. The gradients were collected in fractions and analysed. The number of particles, infectivity titre (in vivo), total protein and viral protein content were evaluated. The purification from infected tissues gave WSSV suspensions with a very high infectivity and an acceptable purity, while virus purified from haemolymph had a high infectivity and a very high purity. Additionally, it was observed that WSSV has an unusually low buoyant density and that it is very sensitive to high external pressures.

Delayed cryopreservation of stallion sperm: effect of iodixanol density gradient centrifugation

Delayed cryopreservation of stallion sperm: effect of iodixanol density gradient centrifugation

Iodixanol density gradient centrifugation for selecting stallion sperm for cold storage and cryopreservation

Density gradient centrifugation can be used for selection of sperm of superior quality and removal of seminal plasma for use in artificial insemination. In this study, the use of two-layer iodixanol density gradient centrifugation was evaluated for processing of stallion semen. The protocol includes centrifugation through a 16% iodixanol top layer of 1.090gmL−1 and collection of motile and intact sperm on a 30% iodixanol bottom layer of 1.165gmL−1. Sperm recovery and effects on sperm quality were determined during cold storage as well as after cryopreservation and compared with ordinary dilution and centrifugation. Two-layer iodixanol density gradient centrifugation allows for selection of greater percentages of morphologically normal and progressively motile sperm compared to ordinary centrifugation. This likely results from collecting sperm on the bottom layer that functions as cushion fluid, which prevents mechanical forces as occur when sperm are packed in a pellet. In addition, percentages of membrane and chromatin integrity are increased when cells are selected based on their density via centrifugation through the top and bottom layers. Removal of seminal plasma and increased initial percentages of motile and membrane intact sperm after iodixanol density gradient centrifugation also result in greater percentages of progressively motile and membrane intact sperm during cold storage as well as after freezing and thawing. In conclusion, the two-layer iodixanol density gradient centrifugation protocol described in this manuscript allows for selection of stallion sperm with greater survival rates for cold storage and cryopreservation.