细胞生物学

Oxidative protein damage is important in various biological processes and age-related diseases. Protein carbonylation is the predominant and most frequently studied form of protein oxidation. It is most frequently detected following its derivatization with 2,4-dinitrophenylhydrazine (DNPH) hapten, followed by its detection with an anti-DNP antibody. However, when used to detect protein carbonylation by western blotting, this method suffers from diminished sensitivity, distortion of protein migration patterns, and unsatisfactory representation of low-abundance proteins. This is due to the poor solubility of DNPH in typical buffer solutions, the acidic protein precipitation due to the use of strong acid for its dissolution, the instability in solution, and the distorted protein migration patterns introduced by an additional salt content generated by the required pH adjustment prior to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). To address the DNPH method limitations, a new Oxime blot technique was developed. This method is based on forming the stable oxime bonds between the protein carbonyl groups and biotin-aminooxy probe in the presence of a p-phenylenediamine (pPDA) catalyst at neutral pH conditions. The derivatization reaction reaches a plateau within 3 h. It ensures efficient and complete derivatization of carbonylated proteins, which are separated by SDS-PAGE without additional manipulation and detected with avidin-HRP and enhanced chemiluminescence (ECL) in western blotting. The Oxime blot protocol allows researchers to reliably and sensitively detect carbonylated proteins and provides a valuable tool for studying oxidative stress in diverse biological settings.

Mitochondria are dynamic organelles with essential roles in energetics and metabolism. Several metabolites are common to both the cytosolic and mitochondrial fractions of the cell. The compartmentalization of metabolites within the mitochondria allows specialized uses for mitochondrial metabolism. Inorganic phosphate (Pi) is one such critical metabolite required for ATP synthesis, via glycolysis and mitochondrial oxidative phosphorylation. Estimating total cellular Pi levels cannot distinguish the distribution of Pi pools across different cellular compartments, such as the cytosol and mitochondria, and therefore separate the contributions made toward glycolysis or other cytosolic metabolic processes vs. mitochondrial outputs. Quantifying Pi pools in mitochondria can therefore be very useful toward understanding mitochondrial metabolism and phosphate homeostasis. Here, we describe a protocol for the fairly rapid, efficient isolation of mitochondria from Saccharomyces cerevisiae by immunoprecipitation for quantitative estimation of mitochondrial and cytosolic Pi pools. This method utilizes magnetic beads to capture FLAG-tagged mitochondria (Tom20-FLAG) from homogenized cell lysates. This method provides a valuable tool to investigate changes in mitochondrial phosphate dynamics. Additionally, this protocol can be coupled with LC–MS approaches to quantitatively estimate mitochondrial metabolites and proteins and can be similarly used to assess other metabolite pools that are partitioned between the cytosol and mitochondria.

Two aconitase isoforms are present in mammalian cells: the mitochondrial aconitase (ACO2) that catalyzes the reversible isomerization of citrate to isocitrate in the citric acid cycle, and the bifunctional cytosolic enzyme (ACO1), which also plays a role as an RNA-binding protein in the regulation of intracellular iron metabolism. Aconitase activities in the different subcellular compartments can be selectively inactivated by different genetic defects, iron depletion, and oxidative or nitrative stress. Aconitase contains a [4Fe-4S]²⁺ cluster that is essential for substrate coordination and catalysis. Many Fe-S clusters are sensitive to oxidative stress, nitrative stress, and reduced iron availability, which forms the basis of redox- and iron-mediated regulation of intermediary metabolism via aconitase and other Fe-S cluster-containing metabolic enzymes, such as succinate dehydrogenase. As such, ACO1 and ACO2 activities can serve as compartment-specific surrogate markers of oxygen levels, reactive oxygen species (ROS), reactive nitrogen species (RNS), iron bioavailability, and the status of intermediary and iron metabolism. Here, we provide a protocol describing a non-denaturing polyacrylamide gel electrophoresis (PAGE)-based procedure that has been successfully used to monitor ACO1 and ACO2 aconitase activities simultaneously in human and mouse cells and tissues.

Macrophages are at the center of innate immunity and iron metabolism. In the case of an infection, macrophages adapt their cellular iron metabolism to deprive iron from invading bacteria to combat intracellular bacterial proliferation. A concise evaluation of the cellular iron content upon an infection with bacterial pathogens and diverse cellular stimuli is necessary to identify underlying mechanisms concerning iron homeostasis in macrophages. For the characterization of cellular iron levels during infection, we established an in vitro infection model where the murine macrophage cell line J774A.1 is infected with Salmonella enterica serovar Typhimurium (S.tm), the mouse counterpart to S. enterica serovar Typhi, under normal and iron-overload conditions using ferric chloride (FeCl₃) treatment. To evaluate the effect of infection and iron stimulation on cellular iron levels, the macrophages are stained with FerroOrange. This fluorescent probe specifically detects Fe²⁺ ions and its fluorescence can be quantified photometrically in a plate reader. Importantly, FerroOrange fluorescence does not increase with chelated iron or other bivalent metal ions. In this protocol, we present a simple and reliable method to quantify cellular Fe²⁺ levels in cultured macrophages by applying a highly specific fluorescence probe (FerroOrange) in a TECAN Spark microplate reader. Compared to already established techniques, our protocol allows assessing cellular iron levels in innate immune cells without the use of radioactive iron isotopes or extensive sample preparation, exposing the cells to stress.

Key features

• Easy quantification of Fe²⁺ in cultured macrophages with a fluorescent probe.

• Analysis of iron in living cells without the need for fixation.

• Performed on a plate reader capable of 540 nm excitation and 585 nm emission by trained employees for handling biosafety level 2 bacteria.

Graphical overview

Macropinocytosis is an evolutionarily conserved process, which is characterized by the formation of membrane ruffles and the uptake of extracellular fluid. We recently demonstrated a role for CYFIP-related Rac1 Interactor (CYRI) proteins in macropinocytosis. High-molecular weight dextran (70kDa or higher) has generally been used as a marker for macropinocytosis because it is too large to fit in smaller endocytic vesicles, such as those of clathrin or caveolin-mediated endocytosis. Through the use of an image-based dextran uptake assay, we showed that cells lacking CYRI proteins internalise less dextran compared to their wild-type counterparts. Here, we will describe a step-by-step experimentation procedure to detect internalised dextran in cultured cells, and an image pipeline to analyse the acquired images, using the open-access software ImageJ/Fiji. This protocol is detailed yet simple and easily adaptable to different treatment conditions, and the analysis can also be automated for improved processing speed.

Reactive oxygen species and reactive nitrogen species (RONS) are involved in programmed cell death in the context of numerous degenerative and chronic diseases. In particular, the ability of cells to maintain redox homeostasis is necessary for an adaptive cellular response to adverse conditions that can cause damage to proteins and DNA, resulting in apoptosis and genetic mutations. Here, we focus on the 2',7'-dichlorodihydrofluorescein diacetate (DCFH₂-DA) assay to detect RONS. Although this fluorescence-based assay is widely utilized due to its high sensitivity to detect changes in cellular redox status that allow measuring alterations in RONS over time, its validity has been a matter of controversy. If correctly carried out, its limitations are understood and results are correctly interpreted, the DCFH₂-DA assay is a valuable tool for cell-based studies.

As one of the main energy metabolism organs, kidney has been proved to have high energy requirements and are more inclined to fatty acid metabolism as the main energy source. Long-chain acyl-CoA dehydrogenases (LCAD) and beta-hydroxyacyl-CoA dehydrogenase (beta-HAD), key enzymes involved in fatty acid oxidation, has been identified as the substrate of acetyltransferase GCN5L1 and deacetylase Sirt3. Acetylation levels of LCAD and beta-HAD regulate its enzymes activity and thus affect fatty acid oxidation rate. Moreover, immunoprecipitation is a key assay for the detection of LCAD and beta-HAD acetylation levels. Here we describe a protocol of immunoprecipitation of acetyl-lysine and western blotting of LCAD and beta-HAD in palmitic acid treated HK-2 cells (human renal tubular epithelial cells). The scheme provides the readers with clear steps so that this method could be applied to detect the acetylation level of various proteins.

Short-chain fatty acids (SCFAs), which are formed mainly by bacteria fermenting undigested carbohydrates in the colon, they are based on the number of carbon atoms in the carbon chain. Organic fatty acids with less than 6 carbon atoms are called short-chain fatty acids. SCFAs are closely related to various aspects of the human body, so more and more researchers concentrate on SCFAs. This protocol describes, a direct injection gas chromatography detection method with a pretreatment method for extracting SCFA from mice feces by combining acidification. The corresponding sample limit of quantization (LOQ) and limit of detection (LOD) are 0.8-1.0 mg/L and 0.5-0.8 mg/L, respectively. The correlation coefficient of calibration curve is greater than 0.999. The recovery rate of the spiked standard is 80%-102%. This method can be used to analyze and determine SCFAs in mice feces. Therefore, this is an economical, effective and reproducible method for SCFAs measurement in mice samples.

Heme oxygenase-1 (HO-1) is a stress responsive enzyme that metabolizes heme and releases free iron, carbon monoxide (CO), and biliverdin (BV), which rapidly undergoes conversion to bilirubin (BL). Estimation of bilirubin is the basis of HO-1 assay. HO-1 activity is widely employed to determine antioxidant response of cells under different physiological stress environment. Intra-macrophage infection often acts as such a stress inducer and measurement of HO-1 activity in infected cells indicates the ability of pathogens towards modulating oxidative response of host. The present protocol describes analysis of HO-1 activity in infected macrophages by spectrophotometric method, which is much less complex and therefore advantageous over other methods like high-performance liquid chromatography, radiochemical methods and detection of CO by gas chromatography. The main steps include: (1) Preparation of macrophage microsomal fraction containing HO-1 (2) Isolation of rat liver cytosolic fraction containing biliverdin reductase and (3) Assessment of heme oxygenase-1 activity by spectrophotometric detection of bilirubin. This method provides a simple and sensitive approach to measure cellular antioxidant response under infected condition.

Cryopreservation is commonly used for the storage of cells, tissues, organs or 3D cell-based products using ultra-low temperatures, which involves the immersion in liquid nitrogen for their long-term preservation. The cryopreservation of several microencapsulated cells is usually performed by the slow freezing with the dimethyl sulfoxide (DMSO) as a cryoprotectant agent (CPA). In this study, we cryopreserved several microencapsulated cells with the natural, non-toxic low molecular-weight hyaluronan (LMW-HA) at 5% and DMSO 10% solution assessing cell viability and metabolic activity after thawing. The cryopreservation of microencapsulated D1 mesenchymal stem cells (D1MSC) and murine myoblast cells (C2C12) with the LMW-HA 5% presented similar outcomes after thawing compared to the DMSO solution, showing the low molecular weight hyaluronan as a natural, non-toxic CPA that can be used preventing the DMSO related adverse effects after the implantation of the cryopreserved cell-based products.