R-DeeP: Proteome-wide and Quantitative Identification of RNA-Dependent Proteins by Density Gradient Ultracentrifugation.

The comprehensive but specific identification of RNA-binding proteins as well as the discovery of RNA-associated protein functions remain major challenges in RNA biology. Here we adapt the concept of RNA dependence, defining a protein as RNA dependent when its interactome depends on RNA. We converted this concept into a proteome-wide, unbiased, and enrichment-free screen called R-DeeP (RNA-dependent proteins), based on density gradient ultracentrifugation. Quantitative mass spectrometry identified 1,784 RNA-dependent proteins, including 537 lacking known links to RNA. Exploiting the quantitative nature of R-DeeP, proteins were classified as not, partially, or completely RNA dependent. R-DeeP identified the transcription factor CTCF as completely RNA dependent, and we uncovered that RNA is required for the CTCF-chromatin association. Additionally, R-DeeP allows reconstruction of protein complexes based on co-segregation. The whole dataset is available at http://R-DeeP.dkfz.de, providing proteome-wide, specific, and quantitative identification of proteins with RNA-dependent interactions and aiming at future functional discovery of RNA-protein complexes.

Virus purification by CsCl density gradient using general centrifugation.

Virus purification by cesium chloride (CsCl) density gradient, which generally requires an expensive ultracentrifuge, is an essential technique in virology. Here, we optimized virus purification by CsCl density gradient using general centrifugation (40,000 × g, 2 h, 4 °C), which showed almost the same purification ability as conventional CsCl density gradient ultracentrifugation (100,000 × g, 1 h, 4 °C) using phages S13' and φEF24C. Moreover, adenovirus strain JM1/1 was also successfully purified by this method. We suggest that general centrifugation can become a less costly alternative to ultracentrifugation for virus purification by CsCl densiy gradient and will thus encourage research in virology.

Effect of rotor type on the separation of isotope-labeled and unlabeled Escherichia coli RNA by isopycnic density ultracentrifugation.

Separation of differentially isotope-labeled bacterial RNA by isopycnic density gradient centrifugation is a critical step in RNA-based stable isotope probing analyses, which help to link the structure and function of complex microbial communities. Using isotope-labeled Escherichia coli RNA, we showed that an 8 mL near-vertical rotor performed better than a 2 mL fixed-angle rotor, thereby corroborating current recommendations. Neither increased concentrations of formamide nor urea in the medium improved the separation results using the fixed-angle rotor.

Methods of isolating extracellular vesicles impact down-stream analyses of their cargoes.

Viable tumor cells actively release vesicles into the peripheral circulation and other biologic fluids, which exhibit proteins and RNAs characteristic of that cell. Our group demonstrated the presence of these extracellular vesicles of tumor origin within the peripheral circulation of cancer patients and proposed their utility for diagnosing the presence of tumors and monitoring their response to therapy in the 1970s. However, it has only been in the past 10 years that these vesicles have garnered interest based on the recognition that they serve as essential vehicles for intercellular communication, are key determinants of the immunosuppressive microenvironment observed in cancer and provide stability to tumor-derived components that can serve as diagnostic biomarkers. To date, the clinical utility of extracellular vesicles has been hampered by issues with nomenclature and methods of isolation. The term "exosomes" was introduced in 1981 to denote any nanometer-sized vesicles released outside the cell and to differentiate them from intracellular vesicles. Based on this original definition, we use "exosomes" as synonymous with "extracellular vesicles." While our original studies used ultracentrifugation to isolate these vesicles, we immediately became aware of the significant impact of the isolation method on the number, type, content and integrity of the vesicles isolated. In this review, we discuss and compare the most commonly utilized methods for purifying exosomes for post-isolation analyses. The exosomes derived from these approaches have been assessed for quantity and quality of specific RNA populations and specific marker proteins. These results suggest that, while each method purifies exosomal material, there are pros and cons of each and there are critical issues linked with centrifugation-based methods, including co-isolation of non-exosomal materials, damage to the vesicle's membrane structure and non-standardized parameters leading to qualitative and quantitative variability. The down-stream analyses of these resulting varying exosomes can yield misleading results and conclusions.

Single-step enrichment of basophils from human peripheral blood by a novel method using a Percoll density gradient.

We have developed a novel continuous flow-through cell separation method using a Percoll density gradient. This method can continuously separate a large number of cells into five fractions according to their densities. To apply this method to the separation of basophils, Percoll density gradients were modified to improve basophil enrichment. When a set of Percoll density gradients was prepared (1.071, 1.075, 1.080, 1.084, and 1.090 g/mL) the basophils in a healthy volunteer were enriched by an average of 23.1 and 63.5% at Percoll densities of 1.075 (fraction 3) and 1.080 g/mL (fraction 4), respectively. On average, the yield of basophils was 1.66 × 10(5) cells in fraction 3 and 1.61 × 10(5) cells in fraction 4 from 9 mL of peripheral blood. The expression of CD203c (cluster of differentiation 203c) on separated basophils was upregulated by anti-immunoglobulin E stimulation similar to basophils in whole blood. Histamine release induced by calcium ionophore was also observed in the separated basophils. The present method will be useful for basophil enrichment since it preserves their function without using counterflow elutriation and immunological reagents, and this method will be effective as a preparative separation for cell purification by flow cytometry.

Excessive centrifugal fields damage high density lipoprotein.

HDL is typically isolated ultracentrifugally at 40,000 rpm or greater, however, such high centrifugal forces are responsible for altering the recovered HDL particle. We demonstrate that this damage to HDL begins at approximately 30,000 rpm and the magnitude of loss increases in a rotor speed-dependent manner. The HDL is affected by elevated ultracentrifugal fields resulting in a lower particle density due to the shedding of associated proteins. To circumvent the alteration of the recovered HDL, we utilize a KBr-containing density gradient and a lowered rotor speed of 15,000 rpm to separate the lipoproteins using a single 96 h centrifugation step. This recovers the HDL at two density ranges; the bulk of the material has a density of about 1.115 g/ml, while lessor amounts of material are recovered at >1.2 g/ml. Thus, demonstrating the isolation of intact HDL is possible utilizing lower centrifuge rotor speeds.

Controllable Assembly and Separation of Colloidal Nanoparticles through a One-Tube Synthesis Based on Density Gradient Centrifugation.

Self-assembly of gold nanoparticles into one-dimensional (1D) nanostructures with finite primary units was achieved by introducing a thin salt (NaCl) solution layer into density gradient before centrifugation. The electrostatic interactions between Au nanoparticles would be affected and cause 1D assembly upon passing through the salt layer. A negatively charged polymer such as poly(acrylic acid) was used as an encapsulation/stabilization layer to help the formation of 1D Au assemblies, which were subsequently sorted according to unit numbers at succeeding separation zones. A centrifugal field was introduced as the external field to overcome the random Brownian motion of NPs and benefit the assembly effect. Such a facile "one-tube synthesis" approach couples assembly and separation in one centrifuge tube by centrifuging once. The method can be tuned by changing the concentration of interference salt layer, encapsulation layer, and centrifugation rate. Furthermore, positively charged fluorescent polymers such as perylenediimide-poly(N,N-diethylaminoethyl methacrylate) could encapsulate the assemblies to give tunable fluorescence properties.

Red blood cell depletion from bone marrow and peripheral blood buffy coat: a comparison of two new and three established technologies.

BACKGROUND: Red blood cell (RBC) depletion is a standard technique for preparation of ABO-incompatible bone marrow transplants (BMTs). Density centrifugation or apheresis are used successfully at clinical scale. The advent of a bone marrow (BM) processing module for the Spectra Optia (Terumo BCT) provided the initiative to formally compare our standard technology, the COBE2991 (Ficoll, manual, "C") with the Spectra Optia BMP (apheresis, semiautomatic, "O"), the Sepax II NeatCell (Ficoll, automatic, "S"), the Miltenyi CliniMACS Prodigy density gradient separation system (Ficoll, automatic, "P"), and manual Ficoll ("M"). C and O handle larger product volumes than S, P, and M. STUDY DESIGN AND METHODS: Technologies were assessed for RBC depletion, target cell (mononuclear cells [MNCs] for buffy coats [BCs], CD34+ cells for BM) recovery, and cost/labor. BC pools were simultaneously purged with C, O, S, and P; five to 18 BM samples were sequentially processed with C, O, S, and M. RESULTS: Mean RBC removal with C was 97% (BCs) or 92% (BM). From both products, O removed 97%, and P, S, and M removed 99% of RBCs. MNC recovery from BC (98% C, 97% O, 65% P, 74% S) or CD34+ cell recovery from BM (92% C, 90% O, 67% S, 70% M) were best with C and O. Polymorphonuclear cells (PMNs) were depleted from BCs by P, S, and C, while O recovered 50% of PMNs. Time savings compared to C or M for all tested technologies are considerable. CONCLUSION: All methods are in principle suitable and can be selected based on sample volume, available technology, and desired product specifications beyond RBC depletion and MNC and/or CD34+ cell recovery.

Semen decontamination for the elimination of seminal HIV-1.

The risk of human immunodeficiency virus (HIV) transmission to the female partner, or potential offspring of an HIV-1 infected man can be reduced using semen decontamination procedures before assisted reproductive treatment (ART). The objective of this study was to determine the efficiency of decontaminating semen samples (n = 186) from 95 HIV-1 sero-positive patients. Aliquots of neat semen were submitted for viral validation by qualitative and quantitative polymerase chain reaction. Semen samples were processed by density gradient centrifugation in combination with a ProInsert™ tube after which aliquots of the processed sperm samples were analysed for the presence of HIV-1. Fifty-four percent of all tested neat semen samples tested positive for HIV-1 DNA, RNA or both (13.4%, 11.3% and 29.0%, respectively). From a total of 103 processed sperm samples that were submitted for viral validation, two samples tested positive for HIV-1 DNA and none for RNA. In conclusion, semen processing with the ProInsert™ followed by viral validation of processed sperm samples should be carried out when providing ART to couples where the male partner is HIV-1 sero-positive.

Enrichment of reticulocytes from whole blood using aqueous multiphase systems of polymers.

This paper demonstrates the enrichment of reticulocytes by centrifuging whole blood through aqueous multiphase systems (AMPSs)-immiscible phases of solutions of polymers that form step-gradients in density. The interfaces of an AMPS concentrate cells; this concentration facilitates the extraction of blood enriched for reticulocytes. AMPS enrich reticulocytes from blood from both healthy and hemochromatosis donors. Varying the osmolality and density of the phases of AMPS provides different levels of enrichment and yield of reticulocytes. A maximum enrichment of reticulocytemia of 64 ± 3% was obtained from donors with hemochromatosis. When used on peripheral blood from normal donors, AMPS can provide a higher yield of enriched reticulocytes and a higher proportion of reticulocytes expressing CD71 than differential centrifugation followed by centrifugation over Percoll. Blood enriched for reticulocytes by AMPS could be useful for research on malaria. Several species of malaria parasites show a preference to invade young erythrocytes and reticulocytes; this preference complicates in vitro cultivation of these species in human blood. Plasmodium knowlesi malaria parasites invade normal human blood enriched for reticulocytes by AMPSs at a rate 2.2 times greater (P < 0.01) than they invade unenriched blood. Parasite invasion in normal blood enriched by AMPS was 1.8 times greater (P < 0.05) than in blood enriched to a similar reticulocytemia by differential centrifugation followed by centrifugation over Percoll. The enrichment of reticulocytes that are invaded by malaria parasites demonstrates that AMPSs can provide a label-free method to enrich cells for biological research.

High precision fractionator for use with density gradient ultracentrifugation.

The recent application of density gradient ultracentrifugation (DGU) for structural sorting of single-walled carbon nanotube samples has created a need for highly selective extraction of closely spaced layers formed in the centrifuged tube. We describe a novel computer-controlled device designed for this purpose. Through the use of fine needles, systematic needle motions, and slow flow rates, multiple sample layers can be aspirated under program control with minimal cross contamination between layers. The fractionator's performance is illustrated with DGU-sorted samples of single-walled carbon nanotubes.

Cell separation by countercurrent centrifugal elutriation: recent developments.

Countercurrent centrifugal elutriation (CCE) is a cell separation technique that separates particles predominantly according to their size, and to some degree according to their specific density, without a need for antibodies or ligands tagging cell surfaces. The principles of this technique have been known for half a century. Still, numerous recent publications confirmed that CCE is a valuable supplement to current cell separation technology. It is mainly applied when homogeneous populations of cells, which mirror an in vivo situation, are required for answering scientific questions or for clinical transplantation, while antibodies or ligands suitable for cell isolation are not available. Currently, new technical developments are expanding its application toward fractionation of healthy and malignant tissue cells and the preparation of dendritic cells for immunotherapy.

Large-scale Ficoll gradient separations using a commercially available, effectively closed, system.

BACKGROUND AIMS: Multiple cell-therapy products require density separation as a part of manufacturing. The traditional method for Ficoll separation, layering cell suspensions over Ficoll in tubes, followed by centrifugation and collection of cells from the interface, is too cumbersome and poses too high a risk of contamination for clinical-scale use. Recently, a system for clinical-scale Ficoll gradient applications has been introduced (Sepax) but this system has limited availability and is costly. METHODS: For preparations of mononuclear cells (MNC) for dendritic cell (DC) production, we developed a Ficoll separation protocol that employs the Haemonetics Cell Saver5 surgical blood salvage and wash instrument. This system uses standard blood bags and tubing, has single-use components, and is effectively closed. We analyzed 37 recent separation processes using this instrument and protocol. We measured depletion of red blood cells (RBC) and polymorphonuclear leukocytes (PMN), and recovery of CD14+ monocytes and MNC. RESULTS: Starting cell counts were 14.6 +/- 8.0 (x10(9)). Total cell recovery was 49.2 +/- 15.2%, RBC depletion was 88.4 +/- 2.8%, PMN depletion was 86.9 +/- 6.1%, MNC recovery was 63.6 +/- 5.0% and CD14+ monocyte recovery was 75.3 +/- 9.9%. CONCLUSIONS: The Cell Saver5 is relatively inexpensive to purchase and use. The instrument and its disposables are licensed by the United States Food and Drug Administration (FDA) for intra-operative blood salvage, and we have obtained approval for investigational use. Our method with this instrument has proven to be simple and efficient for clinical-scale Ficoll separations.

Good manufacturing practice-grade production of unrestricted somatic stem cell from fresh cord blood.

BACKGROUND AIMS: The discovery of unrestricted somatic stem cells (USSC), a non-hematopoietic stem cell population, brought cord blood (CB) to the attention of regenerative medicine for defining more protocols for non-hematopoietic indications. We demonstrate that a reliable and reproducible method for good manufacturing practice (GMP)-conforming generation of USSC is possible that fulfils safety requirements as well as criteria for clinical applications, such as adherence of strict regulations on cell isolation and expansion. METHODS: In order to maintain GMP conformity, the automated cell processing system Sepax (Biosafe) was implemented for mononucleated cell (MNC) separation from fresh CB. After USSC generation, clinical-scale expansion was achieved by multi-layered CellSTACKs (Costar/Corning). Infectious disease markers, pyrogen and endotoxin levels, immunophenotype, potency, genetic stability and sterility of the cell product were evaluated. RESULTS: The MNC isolation and cell cultivation methods used led to safe and reproducible GMP-conforming USSC production while maintaining somatic stem cell character. CONCLUSIONS: Together with implemented in-process controls guaranteeing contamination-free products with adult stem cell character, USSC produced as suggested here may serve as a universal allogeneic stem cell source for future cell treatment and clinical settings.

A Scalable Purification Method for Mitochondria from Trypanosoma brucei.

Due to its unique biology the mitochondrion of Trypanosoma brucei has attracted a lot of interest since many decades, making it arguably the best studied mitochondrion outside yeast and mammals. Here we describe a method allowing purification of mitochondria from procyclic trypanosomes that yields highly enriched and functional organelles. The method is based on isotonic lysis of cells by nitrogen cavitation, DNase I digestion, differential centrifugation and Nycodenz gradient centrifugation. The method is scalable and can be adapted to culture volumes a small as 100 mL or as large as 24 L.

Isolation of Extracellular Vesicles from Leishmania spp.

Exosomes, a class of extracellular vesicles, are released by eukaryotes, bacteria, and archaea, as evident from both in vitro and in vivo studies. These nano-sized double-membraned vesicles play an important role in cell-to-cell communication, dysregulation of the immune system, and pathogenesis in a number of diseases, including leishmaniasis. Leishmania is a genus of obligate intracellular parasites, which infect host macrophages, are transmitted through the bite of a sandfly, and are shown to secrete exosomes with immunomodulatory activities. Given the importance of these vesicles in Leishmania spp. virulence, it is necessary to perform appropriate isolation and characterization in order to further study their relevance in the parasite's infectious life cycle. In this chapter, we describe four methods for the isolation of extracellular vesicles derived from Leishmania species including ultracentrifugation, polyethylene glycol-based precipitation, size-exclusion chromatography, and sucrose-gradient fractionation. Further, we describe the preparation of isolated samples for characterization by nanoparticle tracking analysis, transmission electron microscopy, and proteomic profiling.

Centrifugation of mammalian cells on gradients: a new rotor.

A short-arm rotor increases separation of viable mammalian cells, from mixtures, by low-speed centrifugation; continuous Ficoll density gradients in tissue-culture media are used. We describe the theory and experimental demonstration of the superior separation achieved with this new rotor.

[Erythrocyte removal from bone marrow by density gradient separation using the COBE 2991 cell processor with the triple-bag processing set]. / Désérythrocytation de moelle osseuse par gradient de densité sur COBE 2991 avec le kit triple "bérets".

ABO-incompatible bone marrow transplantation requires red blood cell depletion. Lots of laboratory adopted the technique of density gradient centrifugation (Ficoll-hypaque) using the COBE 2991 cell processor with simple-bag processing set. However, tubing of this set is not adapted to the currently available peristaltic pumps. Moreover, two other sets are required: one for the buffy-coat and one for postgradient cell washing. We developed a method using triple-bag processing set to conduct whole-step procedure (concentration, Ficoll and washing). Peristaltic PVC tubing is provided in one line of the set allowing a safe processing without several connections thus reducing risks of microbial contamination. First, we used buffy-coat of total blood for training, then, we carried out red cell depletion of healthy bone marrow donors. The red blood cell depletion was 97.9+/-1.1% and CD34+ recovery was 89.6+/-8.7%. These results are very close to those obtained with the simple-bag set (red cell depletion.=94.0+/-6.8% and CD34+ recovery=95.9+/-20.3%). We conclude that the triple-bag system, very little used in France, is practical, simplified the manipulation and is more safety than the simple-bag set.

Two-dimensional planar swimming selects for high DNA integrity sperm.

Selection of high-quality sperm is critical to the success of assisted reproductive technologies. Clinical screening for top sperm has long focused on sperm swimming ability when following boundaries or when fully free of constraints. In this work, we demonstrate a sperm selection approach with parallel 2 µm tall confined selection channels that prohibit rotation of the sperm head and require planar swimming. We demonstrate that a planar swimming subpopulation of sperm capable of entering and navigating these channels has DNA integrity superior to the freely-swimming motile or raw sperm populations over a wide range of semen sample qualities. The DNA integrity of the selected sperm was significantly higher than that of the corresponding raw samples for donor samples and clinical patient samples, respectively. In side-by-side testing, this method outperforms current clinical selection methods, density gradient centrifugation and swim-up, as well as sperm selected via general motility. Planar swimming represents a viable sperm selection mechanism with the potential to improve outcomes for couples and offspring.

RNA Stable Isotope Probing (RNA-SIP).

Stable isotope probing is a combined molecular and isotopic technique used to probe the identity and function of uncultivated microorganisms within environmental samples. Employing stable isotopes of common elements such as carbon and nitrogen, RNA-SIP exploits an increase in the buoyant density of RNA caused by the active metabolism and incorporation of heavier mass isotopes into the RNA after cellular utilization of labeled substrates pulsed into the community. Labeled RNAs are subsequently separated from unlabeled RNAs by density gradient centrifugation followed by identification of the RNAs by sequencing. Therefore, RNA stable isotope probing is a culture-independent technique that provides simultaneous information about microbiome community, composition and function. This chapter presents the detailed protocol for performing an RNA-SIP experiment, including the formation, ultracentrifugation, and fractional analyses of stable isotope-labeled RNAs extracted from environmental samples.