The endoplasmic reticulum (ER) is an essential component of the endomembrane system in eukaryotes and plays a crucial role in protein and lipid synthesis, as well as the maintenance of calcium ...homeostasis. Morphologically, the ER is composed primarily of sheets and tubules. The tubular ER is composed of a network of tubular membrane structures, each with diameters ranging from 30 to 50 nanometers. In recent years, there has been in-depth research on the molecular mechanisms of membrane shaping and membrane fusion of the tubular ER. However, there is still limited understanding of the specific physiological functions of the tubular ER. Here, we report a protocol that combines differential centrifugation and immunoprecipitation to specifically enrich microsomes originating from the tubular ER in yeast. The ER tubule-derived microsomes can be further used for proteomic and lipidomic studies or other biochemical analyses.
CsPbX3 perovskite quantum dots (QDs) have attracted increasing attention for the applications of lighting and display due to their excellent properties. Although the preparation of perovskite QDs has ...been widely developed, there is still not a universal and efficient method to purify perovskite QDs, which is due to the labile ionic characteristics of the perovskites. Here, a new purification method is proposed by using the addition of a new antisolvent-dimethicone oil (DMO). Compared to the two commonly used purification methods (direct centrifugation and centrifugation with ethyl acetate antisolvent), the sample purified by DMO addition shows the highest photoluminescence quantum yield (PLQY) and stability. Besides, the yield of the samples obtained from DMO addition is also the highest. This new purification method is also suitable for the products prepared by the room temperature method, where large amounts of QDs with high PLQY can be acquired. Furthermore, a simple differential centrifugation method is designed based on this anti-solvent. Size-selected perovskite QDs with narrow size distribution are obtained from this differential centrifugation method.
•A new efficient and universal method is reported to purify the perovskite quantum dots.•The new purification method can collect almost all of the quantum dots in the crude solution.•The quantum yield of the purified product is higher than the crude solution due to the passivation of dimethicone oil.•A simple differential centrifugation method is designed for the preparation of size-selected perovskite quantum dots.
Microbially induced carbonate precipitation (MICP) is a technique used extensively to address heavy metal pollution but its micro-dynamic process remains rarely explored. In this study, A novel ...Cd-tolerant ureolytic bacterium DL-1 (Pseudochrobactrum sp.) was used to study the micro-dynamic process. With conditions optimized by response surface methodology, the removal efficiency of Cd2+ could achieve 99.89%. Three components were separated and characterized in the reaction mixture of Cd2+ removal by MICP. The quantitative-dynamic distribution of Cd2+ in different components was revealed. Five synergistic effects for Cd2+ removal were found, including co-precipitation, adsorption by precipitation, crystal precipitation on the cell surface, intracellular accumulation and extracellular chemisorption. Importantly, during Cd2+ removal by MICP, the phenomenon that crystalline nanoparticles adhere to the cell surface, but without any micrometer-sized precipitation encapsulated bacterial cells was observed. This indicated that the previously studied model of bacterial cells as nucleation sites for metal cation precipitation and crystal growth is oversimplified. Our findings provided valuable insights into the mechanism of heavy metals removal by MICP, and a more straightforward method for studying biomineralization-related dynamic process.
Extracellular vesicles (EVs) are heterogeneous membranous vesicles secreted by every cell type and offer significant potential in therapy and diagnostics. Differential ultracentrifugation is the gold ...standard for EV isolation, although other techniques including, polyethylene glycol (PEG) precipitation, immunoprecipitation, size exclusion chromatography, and immuno‐isolation approaches are common. Purified EVs can be characterized based on their physical characteristics, biochemical composition, or cell of origin. For size and concentration measurement, nanoparticle tracking analysis (NTA), dynamic light scattering (DLS), and electron microscopy are commonly employed methods. Biochemical analyses of EVs are typically performed using flow cytometry, immunoblotting, or proteomic investigation. Based on tissue of origin, EVs have specific markers that can be used to isolate and purify specific cell‐associated EVs using an affinity selection approach. Despite existence of several methods for isolation and characterization, major limitations associated with each method hinder the progress of the field. Evolving concepts in EV biology possess great promise for better isolation and characterization leading to a better insight of biological function and have immense clinical implications. In this review, we discuss recent advancements in EV isolation and characterization approaches.
The discovery of extracellular vesicles (EVs) has revised the interpretation of intercellular communication. It is now well established that EVs play a significant role in coagulation, inflammation, ...cancer and stem cell renewal and expansion. Their release presents an intriguing, transporting/trafficking network of biologically active molecules, which are able to reach and modulate the function/behavior of the target cells in a variety of ways. Moreover, the presence of EVs in various body fluids points to their potential for use as biomarkers and prognostic indicators in the surveillance/monitoring of a variety of diseases. Although vast knowledge on the subject of EVs has accumulated over the years, there are still fundamental issues associated with the correct approach for their isolation. This review comprises the knowledge on EV isolation techniques that are currently available. The aim of this reveiw was to make both experienced researchers and newcomers to the field aware that different types of EVs require unique isolation approaches. The realization of this 'uniqueness' is the first step in the right direction for the complete assessment of EVs.
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•Starch nanocrystals (SNC) production contained rapid and slow hydrolysis stage.•SNC size reached to nanoscale at hydrolysis of 2 days.•Slow hydrolysis stage mainly involved release ...and modification of SNC.•Differential centrifugation showed good separation on SNC hydrolyzed for 5 days.
Starch nanocrystals (SNCs) were prepared from waxy rice starch via sulfuric acid hydrolysis. The objective focused on the following: i) the hydrolysis kinetics and structural properties of SNCs; ii) the effects of differential centrifugation on the yield and size distribution of SNCs. The hydrolysis was divided into a rapid hydrolysis stage in the initial two days and a slow hydrolysis stage after two days. During the two-day hydrolysis, the average diameter of SNCs reached 244 nm. After two days of hydrolysis, the degree of crystallinity, crystallite size, and melting temperature and enthalpy increased. The proportion of A-branched chains decreased, whereas the proportion of B1-branched chains and molecular weight did not change considerably. Thus, the reaction in the slow hydrolysis stage could be considered as the surface modification and gradual release of SNCs. Furthermore, SNCs with a small size and high charge density could be used for differential centrifugation.
To prevent starch nanocrystals (SNCs) that are generated at an early stage from being hydrolyzed excessively, this study proposed a new separation method, named “neutral dispersion and acidic ...precipitation.” SNCs were prepared from waxy potato starch by sulfuric acid hydrolysis. Based on the results of kinetics and molecular weight, the hydrolysis was divided into three stages, e.g., rapid (initial 1 day), medium (subsequent 1 day) and slow stage (2–5 days). The rapid and medium stages were related to the degradation of amorphous region in starch, and the slow stage mainly referred to SNC release. Therefore, the method was developed to separate SNCs at the slow stage. After centrifugation at 6000 rpm, large particles were removed from the SNC suspension under pH 7. The SNCs with small average size and crystallite size, high relative crystallinity (RC), and high dispersion stability in the supernatant were retained and were then precipitated entirely under pH 5, because pH 5 led to the reduction of dispersion stability of SNCs. Meanwhile, the hydrothermal and dry-thermal stability of separated SNCs were significantly promoted. The separation method has the potential in SNC preparation for increasing the yield and collecting products with small size and high RC.
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•Starch nanocrystals (SNCs) were prepared from waxy potato starch by acid hydrolysis.•The generation of starch nanocrystals mainly occurred in the slow stage.•A method of neutral dispersion and acidic precipitation was used to separate SNCs.•The method avoided excessive hydrolysis of SNCs and reduced the size distribution.•Separated SNCs had high relative crystallinity and thermal stability.
Extracellular vesicles (EVs) from both eukaryotic and prokaryotic cells have been characterized over decades and present many biological properties. Since it has been shown that mycobacterial ...extracellular vesicles (MEVs) of M. ulcerans contain the macrolide toxin mycolactone, MEVs are known to be associated with the pathogenesis of mycobacteria. This chapter describes a method for purifying and characterizing vesicles from in vitro cultures of M. ulcerans. We also describe how purified vesicles can be used in cellular tests, to determine their role in the pathophysiology of M. ulcerans infection.
The research field on extracellular vesicles (EV) has rapidly expanded in recent years due to the therapeutic potential of EV. Adipose tissue human mesenchymal stem cells (ASC) may be a suitable ...source for therapeutic EV. A major limitation in the field is the lack of standardization of the challenging techniques to isolate and characterize EV. The aim of our study was to incorporate new controls for the detection and quantification of EV derived from ASC and to analyze the applicability and limitations of the available techniques. ASC were cultured in medium supplemented with 5% of vesicles-free fetal bovine serum. The EV were isolated from conditioned medium by differential centrifugation with size filtration (0.2 μm). As a control, non-conditioned culture medium was used (control medium). To detect EV, electron microscopy, conventional flow cytometry, and western blot were used. The quantification of the EV was by total protein quantification, ExoELISA immunoassay, and Nanosight. Cytokines and growth factors in the EV samples were measured by multiplex bead array kit. The EV were detected by electron microscope. Total protein measurement was not useful to quantify EV as the control medium showed similar protein contents as the EV samples. The ExoELISA kits had technical troubles and it was not possible to quantify the concentration of exosomes in the samples. The use of Nanosight enabled quantification and size determination of the EV. It is, however, not possible to distinguish protein aggregates from EV with this method. The technologies for quantification and characterization of the EV need to be improved. In addition, we detected protein contaminants in the EV samples, which make it difficult to determine the real effect of EV in experimental models. It will be crucial in the future to optimize design novel methods for purification and characterization of EV.
Studies of the past 15 years have revealed a critical role for extracellular heat shock protein 90alpha (eHsp90α) in the development of several human disorders, including wound healing, cachexia ...(muscle wasting), inflammatory diseases, and cancers. The two established functions of highly purified eHsp90α protein are to promote cell survival and to stimulate cell migration. However, the mechanism of secretion and the method of isolation of eHsp90α remained to be standardized. Among the half a dozen reported methodologies, differential centrifugation is considered the "gold standard" largely for its quantitative recovery of eHsp90α from a conditioned medium of cultured cells. Herein, we describe a revised protocol that isolates three fractions of extracellular vesicles with distinct ranges of diameters and the leftover vesicle-free supernatant for biochemical analyses, especially eHsp90α, from tumor cell-conditioned media. Quantitation of the relative amount of eHsp90α can be carried out with known amounts of recombinant Hsp90α protein on the same SDS-PAGE. We believe that this modified methodology will prove to be a useful tool for studying eHsp90α in cultured cells and beyond.
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