Magnetic Labeling Research Articles

Microfluidic-enabled magnetic labelling of nanovesicles for bioanalytical applications.

Bearing multiple functionalities dramatically increases nanomaterial capabilities to enhance analytical assays by improving sensitivity, selectivity, sample preparation, or signal read-out strategies. Magnetic properties are especially desirable for nanoparticles and nanovesicles as they assist in negating diffusion limitations and improving separation capabilities. Here, we propose a microfluidic method that reliably labels functional nanovesicles while avoiding the risk of crosslinking that would lead to large conglomerates as typically observed in bulk reactions. Thus, the carboxy groups of bi-functional biotinylated fluorescent liposomes were activated in bulk. They were then covalently bound to amino group presenting magnetic beads immobilized through a magnetic field within microfluidic channels. Microfluidic design and coupling strategy optimization led to a 62% coupling efficiency when using 1 μm magnetic beads. The yield dropped to 13% with 30 nm magnetic nanoparticles (MNPs) likely due to crowding of the MNPs on the magnet. Finally, both populations of these tri-functional liposomes were applied to a biological binding assay demonstrating their superior performance under the influence of a magnetic field. The microfluidic functionalization strategy lends itself well for massively parallelized production of larger volumes and can be applied to micro- and nanosized vesicles and particles.

Microfluidic chip for graduated magnetic separation of circulating tumor cells by their epithelial cell adhesion molecule expression and magnetic nanoparticle binding

The enumeration of circulating tumor cells (CTCs) in the peripheral bloodstream of metastatic cancer patients has contributed to improvements in prognosis and therapeutics. There have been numerous approaches to capture and counting of CTCs. However, CTCs have potential information beyond simple enumeration and hold promise as a liquid biopsy for cancer and a pathway for personalized cancer therapy by detecting the subset of CTCs having the highest metastatic potential. There is evidence that epithelial cell adhesion molecule (EpCAM) expression level distinguishes these highly metastatic CTCs. The few previous approaches to selective CTC capture according to EpCAM expression level are reviewed. A new two-stage microfluidic device for separation, enrichment and release of CTCs into subpopulations sorted by EpCAM expression level is presented here. It relies upon immunospecific magnetic nanoparticle labeling of CTCs followed by their field- and flow-based separation in the first stage and capture as discrete subpopulations in the second stage. To fine tune the separation, the magnetic field profile across the first stage microfluidic channel may be modified by bonding small Vanadium Permendur strips to its outer walls. Mathematical modeling of magnetic fields and fluid flows supports the soundness of the design.

A pictorial review of brain arterial spin labelling artefacts and their potential remedies in clinical studies.

Arterial spin labelling is an emerging non-invasive magnetic resonance imaging technique for estimating the cerebral perfusion without the requirement for gadolinium-based intravenous contrast agents. Despite the wide range of applications in epilepsy, dementia, brain tumours, vascular malformations and stroke imaging, obtaining clinically useful arterial spin labelling data is technically challenging and prone to numerous artefacts. The objective of this review is to provide a comprehensive pictorial overview of the various artefacts associated with arterial spin labelling, particularly three-dimensional fast spin echo pseudocontinuous arterial spin labelling with spiral readout. These artefacts could be broadly classified as those occurring during the magnetic labelling, arterial transit or image acquisition. Arterial spin labelling artefacts of clinical diagnostic utility are also elaborated. A thorough knowledge of the basis of these artefacts will avoid diagnostic pitfalls while interpreting arterial spin labelling images. Important tips to reduce or overcome these artefacts are also discussed.

Saccharide-capped Superparamagnetic Copper Cations-doped Magnetite Nanoparticles for Biomedical Applications: A Novel and Simple Synthesis Procedure, In-situ Surface Engineering and Characterization

Background: Recently, superparamagnetic and electromagnetic nano-materials have been extensively studied and their potential applications have also been investigated in various fields. In this regard, currently, Fe3O4 NPs are valuable candidates as diagnostic agents such as magnetic resonance imaging, enzyme immobilization, biosensing and cell labeling, and therapeutic probes, including drug delivery, bacteria detection, magnetic separation, and hyperthermia agents. Objective: In this study, electrochemical synthesis of Cu2+ cations-doped superparamagnetic magnetite nanoparticles (Cu-SMNPs) and their in situ surface coating with saccharides (i.e., glucose, sucrose and starch) are reported. The prepared glucose/Cu-SMNPs, sucrose/Cu-SMNPs and starch/Cu-SMNPs samples are characterized by structural, magnetic and morphological analyses by XRD, FT-IR, FE-SEM, EDAX and VSM. The suitability of the prepared samples for biomedical use is also proved. Methods: A simple cathodic electrochemical set-up was used to fabricate the iron oxide samples. The bath electrolyte was one litre deionized water containing 1.5g iron chloride, 3g iron nitrate, 0.5g copper chloride and 0.5g saccharide (i.e., glucose or sucrose or starch). The cathode and anode electrodes were connected to a DC power supply (PROVA 8000) as the power source. The deposition experiments were conducted at 10 mA cm-2 for 30 min. For the preparation of glucose/Cu-SMNPs, sucrose/Cu-SMNPs and starch/Cu-SMNPs samples, three electrodeposition experiments were carried out in three similar baths with only a change in the dissolved saccharide type. The prepared SMNPs samples were characterized by structural, morphological and magnetic analyses including X-ray powder diffraction (XRD, a Phillips PW-1800 diffractometer Smart Lab), field-emission scanning electron microscopy (FE-SEM, Mira 3-XMU with accelerating voltage of 100 kV), transmission electron microscopy (TEM, model Zeiss EM900 with an accelerating voltage of 80 kV), fourier transform infrared (FT-IR, a Bruker Vector 22 Fourier transformed infrared spectrometer) and vibrating sample magnetometers (VSM, model Lakeshore 7410). Results: Three types of metal-cations doped superparamagnetic magnetite nanoparticles (SMNPs), glucosegrafted Cu2+-doped MNPs (glucose/Cu-SMNPs), sucrose-grafted Cu2+-doped SMNPs (sucrose/Cu-SMNPs) and starch-grafted Cu2+-doped SMNPs (starch/Cu-SMNPs), were prepared for the first time. Fourier-transform infrared spectroscopy, field-emission scanning electron microscopy and energy dispersive X-ray techniques proved the presence of saccharide capped layer on the surface of deposited SMNPs and also copper cations doping on their crystal structures. Superparamagnetic behaviors, including low coercivity and remanence values, were observed for all the prepared samples. Conclusion: SMNPs capped with saccharides (i.e., glucose, sucrose and starch) were successfully synthesized via one-pot simple deposition procedures. These particles showed suitable superparamagnetic properties with negligible remanence values and proper saturation magnetization, thus proving that they all have required physicochemical and magnetic characteristics for biomedical purposes.

3D Magnetic Alignment of Cardiac Cells in Hydrogels.

Tissue engineering aims to repair or replace deficient tissue by delivering constructs that mimic the native in vivo structure. One challenge in cardiac tissue engineering approaches is to achieve intrinsic cardiac organization, particularly the alignment of cardiomyocytes. Here, we propose a strategy for 3D manipulation and alignment of cardiomyocytes by combining magnetism and a hydrogel. The advantage of using magnetic forces is that they act remotely on the cells when these are endowed with magnetization via the internalization of magnetic nanoparticles. The magnetic actuation then allows obtaining, almost instantaneously and before gel transition, an aligned biomimetic cardiac tissue construct. Gel transition enables us to keep the cellular pattern once the magnetic field was removed. This cardiac tissue engineering approach was tested with both H9c2 cell line and primary cardiomyocytes, and with both a synthetic hydrogel and a natural one, Pluronic F-127 and fibrin, respectively. Key parameters of the anisotropic tissue formation were assessed. Hydrogel rheology is provided, and the impact of cell density and magnetic labeling on cell-cell alignment is assessed. Immunofluorescence confirms the presence of several cardiac markers upon chaining, demonstrating the functionality of the tissue-like cell alignment obtained via magnetic actuation.

MAGNETOPHORETIC CIRCUIT BIOCOMPATIBILITY

Recently, we introduced magnetophoretic circuits, composed of overlaid magnetic and metallic layers, as a novel single-cell analysis (SCA) tool. We showed the ability of these circuits in organizing large single-particle and particle-pair arrays. Assembling the cells in microarrays is performed with the ultimate goal of running temporal phenotypic analyses. However, for long-term studies, a suitable microenvironment for the cells to normally grow and differentiate is needed. Towards this goal, in this study, we run required biocompatibility tests, based on which we make the magnetophoretic-based microchip a suitable home for the cells to grow. The results confirm the ability of these chips in cell handling and show no unwanted cell behavior alteration due to the applied shear stress on them, the magnetic labeling, or the microenvironment. After this achievement, this tool would be ready for running important single-cell studies in oncology, virology, and medicine.

Abstract 1592: A complete workflow for the isolation of tumor-infiltrating leukocytes from human tumors and humanized mouse models

Abstract The in-depth characterization of tumor-infiltrating leukocytes (TILs) is crucial to further improve cancer immunotherapies. Since TILs generally constitute a small subpopulation of solid tumors, they can be lost in the background noise of downstream analyses such as flow cytometry or single cell sequencing. Therefore, improving pre-enrichment methods for TILs is necessary to increase the sensitivity of such studies and save resources spent on the analysis of contaminating cell populations. We have established a reliable workflow combining automated tissue dissociation with positive selection of CD45+ TILs from human tumors as well as humanized mouse models. Human tumor samples were stored and shipped in MACS® Tissue Storage Solution, maintaining cell viability and phenotype up to 48 hours after collection. Tumor dissociation was automated using the gentleMACS™Octo Dissociator and optimized for epitope conservation to overcome bias in immune-phenotyping caused by dissociation with aggressive or impure enzymes. The direct isolation of TILs by MACS® Technology was optimized for the characteristics of solid tumors, such as the presence of debris and dead cells, based on a CD45-specific enrichment reagent. As an example, TIL isolation from a human colorectal carcinoma led to purities of target cells >92% and yields >80%. To allow for the isolation of label free TILs, we developed an isolation strategy based on the REAlease™ Technology. Again, this method is independent of the tumor entity and even tumors with low frequencies of TIL infiltration, such as an ovarian carcinoma with a frequency of 4%, could be enriched to >90% purity. Upon Bead release, >99% of the cells showed no residual magnetic labeling, hence TILs could be subsequently used for a second round of magnetic isolation for the isolation of multiple subpopulations. Additionally, we demonstrated that the REAlease Biotin Complex was effectively removed from the target cells as well, making them non-distinguishable from non-labeled cells before separation. Due to the removal of all labels, epitopes used for isolation are completely available for re-labeling and can be further used for any downstream application. Taken together, we have developed a reliable workflow for the dissociation of solid tumors followed by the isolation of TILs in less than 90 minutes. We could show efficient separation performance using the standard MACS® as well as REAlease™ Technology for positive isolation of TILs. The REAlease™ Technology allows for the efficient removal of MicroBeads and the entire labeling complex, resulting in cells that are suited for a second magnetic labeling step or any kind of downstream analysis. Citation Format: Janina Brauner, Wa'el Al Rawashdeh, Andreas Bosio, Olaf Thorsten Hardt. A complete workflow for the isolation of tumor-infiltrating leukocytes from human tumors and humanized mouse models [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1592.

Abstract LB-024: Development of a non-invasive clinically applicable real time cell tracking platform for evaluating cell-based cancer therapy using magnetic resonance imaging (MRI)

Abstract While advancement in chimeric antigen receptors (CARs) has greatly improved the anti-tumor activity of immune cell-based therapies utilizing modified T lymphocytes (CAR-T) and natural killer cells (CAR-NK), there remains a critical unmet need for diagnostic technologies to help evaluate their biological performance effectively and efficiently after injecting into the body.The ability to better understand the biodistribution of injected cells and devise dosing strategies are key determinants to the success or failure of cell therapy. However, clinicians are limited by the available technology for real time in-vivo tracking of cell therapy products in clinical settings. Here we present a clinically applicable novel magnetic agent that can effectively label a variety of cell types used in cancer cell therapy development including T cells, NK cells, and neural stem cells (NSCs), and when combined with the 3D ultrashort echo time Cones (3D UTE-Cones) sequence, can enable quantitative assessment of cell therapy products using MRI. In this study, we optimized loading of our tracking agent into T cells, NK cells, and NSCs, evaluated the morphology and viability of the labeled cells, and confirmed internalization of the labeling agent by Prussian blue staining and TEM. The in vitro MRI properties of the labeled cells were quantitated by the 3D UTE-Cones sequence. Ex vivo MRI analysis was performed by injecting labeled cells into harvested mouse livers and brains to ascertain their MRI visibility in tissues using a clinical MRI scanner. We confirmed that our magnetic labeling agent efficiently labeled each cell line tested, and the morphology or viability of the labeled cells were unaffected. The MRI detectability of labeled cells were confirmed by in vitro MRI phantoms. Further, using quantitative susceptibility mapping (QSM), T1, and T2* mapping based on 3D UTE-Cones sequences, we found near perfect correlations between cell density and MRI properties. By applying the parameters established in the MRI phantoms, we were able to detect and quantity the number of labeled cells injected into mouse livers and brains ex vivo. In summary, we have developed a clinically applicable imaging-based platform that empowers cell therapy researchers to better understand the results of their biodistribution and dosing studies through real-time cell tracking and quantitative assessments using MRI. Citation Format: Johnny C. Akers, Zhao Wei, Hyungseok Jang, Eric Chang, Jiang Du, Mya S. Thu. Development of a non-invasive clinically applicable real time cell tracking platform for evaluating cell-based cancer therapy using magnetic resonance imaging (MRI) [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr LB-024.

Regional cortical thinning and cerebral hypoperfusion in patients with panic disorder

BackgroundAlthough recent neuroimaging studies have suggested that functional deficits in facial perception are associated with panic disorder (PD), the possibility of cortical thickness and perfusion abnormalities have not been studied in patients with PD. We aimed to investigate alterations in cortical thickness and regional cerebral blood flow (CBF) between PD patients and healthy controls (HCs) using three-dimensional (3D) T1-weighted magnetic resonance imagery (MRI) and 3D arterial spin labeling (ASL) perfusion MRI. MethodAn automated surface-based method (Cat12) measured the cortical thickness of each subject. Z-score normalization for CBF maps was used to generate Z-score maps. Statistical comparisons were performed using statistical parametric mapping with two-sample t-tests. ResultsSubjects with PD, unlike HCs, displayed cortical thinning in the right fusiform gyrus (FG). Post hoc analysis also revealed a decreased Z-score in the right FG. There was significant positive correlation between the Z-score and the cortical thickness of the right FG. The cortical thickness and Z-score were negatively correlated with the Panic Disorder Severity Scale and Hamilton Rating Scale for Anxiety scores. LimitationsThe small sample size may have restricted the identification of additional differences. Other caveats included the use of medication by nine participants. ConclusionsThese results provide further evidence of the significant role structural and functional deficits in the right FG play in patients with PD. Due to the observed regional specificity, this finding bears important clinical implications for potential treatment strategies.

Tracking the expression of therapeutic protein targets in rare cells by antibody-mediated nanoparticle labelling and magnetic sorting.

Molecular-level features of tumours can be tracked using single-cell analyses of circulating tumour cells (CTCs). However, single-cell measurements of protein expression for rare CTCs are hampered by the presence of a large number of non-target cells. Here, we show that antibody-mediated labelling of intracellular proteins in the nucleus, mitochondria and cytoplasm of human cells with magnetic nanoparticles enables analysis of target proteins at the single-cell level by sorting the cells according to their nanoparticle content in a microfluidic device with cell-capture zones sandwiched between arrays of magnets. We used the magnetic labelling and cell-sorting approach to track the expression of therapeutic protein targets in CTCs isolated from blood samples of mice with orthotopic prostate xenografts and from patients with metastatic castration-resistant prostate cancer. We also show that mutated proteins that are drug targets or markers of therapeutic response can be directly identified in CTCs, analysed at the single-cell level and used to predict how mice with drug-susceptible and drug-resistant pancreatic tumour xenografts respond to therapy.

Carbon-Coated Superparamagnetic Nanoflowers for Biosensors Based on Lateral Flow Immunoassays.

Superparamagnetic iron oxide nanoflowers coated by a black carbon layer (Fe3O4@C) were studied as labels in lateral flow immunoassays. They were synthesized by a one-pot solvothermal route, and they were characterized (size, morphology, chemical composition, and magnetic properties). They consist of several superparamagnetic cores embedded in a carbon coating holding carboxylic groups adequate for bioconjugation. Their multi-core structure is especially efficient for magnetic separation while keeping suitable magnetic properties and appropriate size for immunoassay reporters. Their functionality was tested with a model system based on the biotin–neutravidin interaction. For this, the nanoparticles were conjugated to neutravidin using the carbodiimide chemistry, and the lateral flow immunoassay was carried out with a biotin test line. Quantification was achieved with both an inductive magnetic sensor and a reflectance reader. In order to further investigate the quantifying capacity of the Fe3O4@C nanoflowers, the magnetic lateral flow immunoassay was tested as a detection system for extracellular vesicles (EVs), a novel source of biomarkers with interest for liquid biopsy. A clear correlation between the extracellular vesicle concentration and the signal proved the potential of the nanoflowers as quantifying labels. The limit of detection in a rapid test for EVs was lower than the values reported before for other magnetic nanoparticle labels in the working range 0–3 × 107 EVs/μL. The method showed a reproducibility (RSD) of 3% (n = 3). The lateral flow immunoassay (LFIA) rapid test developed in this work yielded to satisfactory results for EVs quantification by using a precipitation kit and also directly in plasma samples. Besides, these Fe3O4@C nanoparticles are easy to concentrate by means of a magnet, and this feature makes them promising candidates to further reduce the limit of detection.

Label-Free Sensitive Detection of Steroid Hormone Cortisol Based on Target-Induced Fluorescence Quenching of Quantum Dots.

We discovered that several types of steroid hormones quench the fluorescence of quantum dots (QDs) at close proximity. Inspired by the finding, we developed a new type of biosensor for the sensitive detection of cortisol via direct fluorescence quenching of functionalized QD probes directly induced by the capture of target cortisol without additional reporter reagents. The detection selectivity was provided by cortisol-selective aptamers or anticortisol antibodies conjugated on the QD surfaces. With the magnetic nanoparticle labeling, the new sensing method enabled rapid cortisol sensing at physiologically relevant concentrations and yielded the detection limit of ∼1 nM for aptamer-based sensors and ∼100 pM for antibody-based sensors. We also evaluated the new detection method using saliva samples with an optimized sample preparation process under the assistance of magnetic manipulation. The result showed a satisfying recovery rate for spiked saliva tests. The facile sensing technology offers an appealing approach for the detection of steroid hormones in point-of-care settings.

On-Chip Sonoporation-Based Flow Cytometric Magnetic Labeling.

Tracing magnetically labeled cells with magnetic resonance imaging (MRI) is an emerging and promising approach to uncover in vivo behaviors of cells in cell therapy. Today, existing methods for the magnetic labeling of cells are cumbersome and time-consuming, which has greatly limited the progress of such studies on cell therapy. Thus, in this study, using the flow cytometric loading technology, we develop a sonoporation-based microfluidic chip (i.e., a microfluidic chip integrated with ultrasound; MCU), to achieve the safe, instant, convenient, and continuous magnetic labeling of cells. For the MCU we designed, a suitable group of operating conditions for safely and efficiently loading superparamagnetic iron oxide (SPIO) nanoparticles into DC2.4 cells was identified experimentally. Under the identified operating conditions, the DC2.4 cells could be labeled in approximately 2 min with high viability (94%) and a high labeling quantity of SPIO nanoparticles (19 pg of iron per cell). In addition, the proliferative functions of the cells were also well maintained after labeling. Furthermore, the in vivo imaging ability of the DC2.4 cells labeled using the MCU was verified by injecting the labeled cells into the leg muscle of the C57BL/6 mice. The results show that the excellent imaging outcome can be continuously achieved for 7 days at a density of 106 cells/mL. This work can provide insight for the design of magnetic cell labeling devices and promote the MRI-based study of cell therapies.

Effect of medium and aggregation on antibacterial activity of nanodiamonds

Fluorescent nanodiamonds are widely used as abrasives, optical or magnetic labels, in drug delivery or nanoscale sensing. They are considered very biocompatible in mammalian cells. However, in bacteria the situation looks different and results are highly controversial. This article presents a short review of the published literature and a systematic experimental study of different strains, nanoparticle sizes and surface chemistries. Most notably, particle aggregation behaviour and bacterial clumping are taken into consideration to explain reduced colony counts, which can be wrongly interpreted as a bactericidal effect. The experiments show no mechanism can be linked to a specific material property, but prove that aggregation and bacteriostatic effect of nanodiamond attachment play a significant role in the reported results.

L-Lysine-modified Fe3O4 nanoparticles for magnetic cell labeling

The efficiency of magnetic labeling with L-Lys-modified Fe3O4 magnetic nanoparticles (MNPs) and the stability of magnetization of rat adipose-derived mesenchymal stem cells, lineage-negative (Lin(–)) hematopoietic progenitor cells from mouse bone marrow and human leukemia K562 cells were studied. For this purpose, covalent modification of MNPs with 3-aminopropylsilane and N-di-Fmoc-L-lysine followed by removal of N-protecting groups was carried out. Since the degree of hydroxylation of the surface of the starting nanoparticles plays a crucial role in the silanization reaction and the possibility of obtaining stable colloidal solutions. In present work we for the first time performed a comparative qualitative and quantitative evaluation of the number of adsorbed water molecules and hydroxyl groups on the surface of chemically and physically obtained Fe3O4 MNPs using comprehensive FTIR spectroscopy and thermogravimetric analysis. The results obtained can be further used for magnetic labeling of cells in experiments in vitro and in vivo.

Magnetosomes for bioassays by merging fluorescent liposomes and magnetic nanoparticles: encapsulation and bilayer insertion strategies

Magnetized liposome (magnetosomes) labels can overcome diffusion limitations in bioassays through fast and easy magnetic attraction. Our aim therefore was to advance the understanding of factors influencing their synthesis focusing on encapsulation strategies and synthesis parameters. Magnetosome synthesis is governed by the surface chemistry and the size of the magnetic nanoparticles used. We therefore studied the two possible magnetic labelling strategies, which are the incorporation of small, hydrophobic magnetic nanoparticles (MNPs) into the bilayer core (b-liposomes) and the entrapment of larger hydrophilic MNPs into the liposomes’ inner cavity (i-liposomes). Furthermore, they were optimized and compared for application in a DNA bioassay. The major obstacles observed for each of these strategies were on the one hand the need for highly concentrated hydrophilic MNPs, which is limited by their colloidal stability and costs, and on the other hand the balancing of magnetic strength vs. size for the hydrophobic MNPs. In the end, both strategies yielded magnetosomes with good performance, which improved the limit of detection of a non-magnetic DNA hybridization assay by a factor of 3–8-fold. Here, i-liposomes with a magnetization yield of 5% could be further improved through a simple magnetic pre-concentration step and provided in the end an 8-fold improvement of the limit of detection compared with non-magnetic conditions. In the case of b-liposomes, Janus-like particles were generated during the synthesis and yielded a fraction of 15% magnetosomes directly. Surprisingly, further magnetic pre-concentration did not improve their bioassay performance. It is thus assumed that magnetosomes pull normal liposomes through the magnetic field towards the surface and the presence of more magnetosomes is not needed. The overall stability of magnetosomes during storage and magnetic action, their superior bioassay performance, and their adaptability towards size and surface chemistry of MNPs makes them highly valuable signal enhancers in bioanalysis and potential tools for bioseparations.Graphical abstract

Intravenous administration of iPS-MSCSPIONs mobilized into CKD parenchyma and effectively preserved residual renal function in CKD rat.

This study traced intravenously administered induced pluripotent stem cell (iPSC)‐derived mesenchymal stem cells (MSC) and assessed the impact of iPSC‐MSC on preserving renal function in SD rat after 5/6 nephrectomy. The results of in vitro study showed that FeraTrack™Direct contrast particles (ie intracellular magnetic labelling) in the iPSC‐MSC (ie iPS‐MSCSPIONs) were clearly identified by Prussian blue stain. Adult‐male SD rats (n = 40) were categorized into group 1 (SC), group 2 [SC + iPS‐MSCSPIONs (1.0 × 106cells)/intravenous administration post‐day‐14 CKD procedure], group 3 (CKD), group 4 [CKD + iPS‐MSCSPIONs (0.5 × 106cells)] and group 5 [CKD + iPS‐MSCSPIONs (1.0 × 106cells)]. By day‐15 after CKD induction, abdominal MRI demonstrated that iPS‐MSCSPIONs were only in the CKD parenchyma of groups 4 and 5. By day 60, the creatinine level/ratio of urine protein to urine creatinine/kidney injury score (by haematoxylin and eosin stain)/fibrotic area (Masson's trichrome stain)/IF microscopic finding of kidney injury molecule‐1 expression was lowest in groups 1 and 2, highest in group 3, and significantly higher in group 4 than in group 5, whereas IF microscopic findings of podocyte components (ZO‐1/synaptopodin) and protein levels of anti‐apoptosis ((Bad/Bcl‐xL/Bcl‐2) exhibited an opposite pattern to creatinine level among the five groups (all P < .0001). The protein expressions of cell‐proliferation signals (PI3K/p‐Akt/m‐TOR, p‐ERK1/2, FOXO1/GSK3β/p90RSK), apoptotic/DNA‐damage (Bax/caspases8‐10/cytosolic‐mitochondria) and inflammatory (TNF‐α/TNFR1/TRAF2/NF‐κB) biomarkers displayed an identical pattern to creatinine level among the five groups (all P < .0001). The iPS‐MSCSPIONs that were identified only in CKD parenchyma effectively protected the kidney against CKD injury.

Sorting and magnetic-based isolation of reticulated platelets from peripheral blood

Reticulated Platelets (RPs) are large, RNA-rich, prothrombotic and hyperactive platelets known to be elevated in high-risk populations such as diabetics and patients with acute coronary syndrome. High levels of RPs correlate with mortality and adverse cardiovascular events in patients with coronary artery disease as well as with an insufficient antiplatelet response to thienopyridines and aspirin after percutaneous coronary interventions, making them an appealing drug target. However, processing of platelets is challenging and no specific marker for RPs exists. Until now, the gold standard laboratory-based method to study them is based on the flow cytometric measurement of their cell size and their RNA-content with the fluorescent dye Thiazole Orange (TO). Nevertheless, standardized protocols for staining and processing of RPs are missing and the existing techniques were not applied for cell sorting. We provide here a structured and reproducible method to detect, isolate and collect RPs from peripheral blood by RNA-specific staining with TO implementing several platelet inhibitors as well as magnetic labeling allowing sufficient cell recovery and deep biological investigation of these platelets.

Rapid Ultrasensitive Gel-Free Immunoblotting with Magnetic Labels

Immunoblotting is widely used for the detection of proteins using specific antibodies. We present here a new immunoblotting method, which is characterized by exceptional sensitivity, rapidness, and low consumption of antibodies. A thin conductive layer between touching hydrophilic cellulose membranes instead of polyacrylamide gel is used for the electrophoretic separation of proteins. Contrary to common Western blotting, the separation occurs in nondenaturing conditions. The membrane surface is smoothed by deposition of the cellulose layer and modified with azidophenyl groups, allowing for the photochemical in situ immobilization of proteins, which are carried out after the electrophoresis. Thus, the additional step of transferring the protein from the gel onto the membrane is eliminated. Specific protein bands are then visualized by decoration with magnetic beads. The limit of detection of interleukin IL-1β reaches 0.3 fg or ∼104 molecules, whereas the total blotting time is about 5 min. The application of the technique is demonstrated by the detection of IL-1β, total IgA, and IgA specific to Mycobacterium tuberculosis antigen in the exhaled breath samples, obtained from healthy subjects and tuberculosis patients.

Objective Pain Assessment: a Key for the Management of Chronic Pain.

The individual and social burdens associated with chronic pain have been escalating globally. Accurate pain measurement facilitates early diagnosis, disease progression monitoring and therapeutic efficacy evaluation, thus is a key for the management of chronic pain. Although the “golden standards” of pain measurement are self-reported scales in clinical practice, the reliability of these subjective methods could be easily affected by patients’ physiological and psychological status, as well as the assessors’ predispositions. Therefore, objective pain assessment has attracted substantial attention recently. Previous studies of functional magnetic resonance imaging (fMRI) revealed that certain cortices and subcortical areas are commonly activated in subjects suffering from pain. Dynamic pain connectome analysis also found various alterations of neural network connectivity that are correlated with the severity of clinical pain symptoms. Electroencephalograph (EEG) demonstrated suppressed spontaneous oscillations during pain experience. Spectral power and coherence analysis of EEG also identified signatures of different types of chronic pain. Furthermore, fMRI and EEG can visualize objective brain activities modulated by analgesics in a mechanism-based way, thus bridge the gaps between animal studies and clinical trials. Using fMRI and EEG, researchers are able to predict therapeutic efficacy and identify personalized optimal first-line regimens. In the future, the emergence of magnetic resonance spectroscopy and cell labelling in MRI would encourage the investigation on metabolic and cellular pain biomarkers. The incorporation of machine learning algorithms with neuroimaging or behavior analysis could further enhance the specificity and accuracy of objective pain assessments.