Probing cell proliferation: Considerations for dye selection.

Broadly speaking, cell tracking dyes are fluorescent compounds that bind stably to components on or within the cells so the fate of the labeled cells can be followed. Their staining should be bright and homogeneous without affecting cell function. For purposes of monitoring cell proliferation, each time a cell divides the intensity of cell tracking dye should diminish equally between daughter cells. These dyes can be grouped into two different classes. Protein reactive dyes label cells by reacting covalently but non-selectively with intracellular proteins. Carboxyfluorescein diacetate succinimidyl ester (CFSE) is the prototypic general protein label. Membrane intercalating dyes label cells by partitioning non-selectively and non-covalently within the plasma membrane. The PKH membrane dyes are examples of lipophilic compounds whose chemistry allows for their retention within biological membranes without affecting cellular growth, viability, or proliferation when used properly. Here we provide considerations based for labeling cell lines and peripheral blood mononuclear cells using both classes of dyes. Examples from optimization experiments are presented along with critical aspects of the staining procedures to help mitigate common risks. Of note, we present data where a logarithmically growing cell line is labeled with both a protein dye and a membrane tracking dye to compare dye loss rates over 6days. We found that dual stained cells paralleled dye loss of the corresponding single stained cells. The decrease in fluorescence intensity by protein reactive dyes, however, was more rapid than that with the membrane reactive dyes, indicating the presence of additional division-independent dye loss.

Tuning the double lipidation of salmon calcitonin to introduce a pore-like membrane translocation mechanism.

A widespread strategy to increase the transport of therapeutic peptides across cellular membranes has been to attach lipid moieties to the peptide backbone (lipidation) to enhance their intrinsic membrane interaction. Efforts in vitro and in vivo investigating the correlation between lipidation characteristics and peptide membrane translocation efficiency have traditionally relied on end-point read-out assays and trial-and-error-based optimization strategies. Consequently, the molecular details of how therapeutic peptide lipidation affects it's membrane permeation and translocation mechanisms remain unresolved. Here we employed salmon calcitonin as a model therapeutic peptide and synthesized nine double lipidated analogs with varying lipid chain lengths. We used single giant unilamellar vesicle (GUV) calcein influx time-lapse fluorescence microscopy to determine how tuning the lipidation length can lead to an All-or-None GUV filling mechanism, indicative of a peptide mediated pore formation. Finally, we used a GUVs-containing-inner-GUVs assay to demonstrate that only peptide analogs capable of inducing pore formation show efficient membrane translocation. Our data provided the first mechanistic details on how therapeutic peptide lipidation affects their membrane perturbation mechanism and demonstrated that fine-tuning lipidation parameters could induce an intrinsic pore-forming capability. These insights and the microscopy based workflow introduced for investigating structure-function relations could be pivotal for optimizing future peptide design strategies.

Simultaneous detection of breast cancer biomarkers circROBO1 and BRCA1 based on a CRISPR-Cas13a/Cas12a system.

Breast cancer is reported to be one of the most lethal cancers in women, and its multi-target detection can help improve the accuracy of diagnosis. In this work, a cluster regularly interspaced short palindromic repeats (CRISPR)-Cas13a/Cas12a-based system was established for the simultaneous fluorescence detection of breast cancer biomarkers circROBO1 and BRCA1. CRISPR-Cas13a and CRISPR-Cas12a were directly activated by their respective targets, resulting in the cleavage of short RNA and DNA reporters, respectively, thus the signals of 6-carboxyfluorescein (FAM) and 6-carboxy-xrhodamine (ROX) were restored. As the fluorescence intensities of FAM and ROX were dependent on the concentrations of circROBO1 and BRCA1, respectively, synchronous fluorescence scanning could achieve one-step detection of circROBO1 and BRCA1 with detection limits of 0.013 pM and 0.26 pM, respectively. The system was highly sensitive and specific, holding high diagnostic potential for the detection of clinical samples. Furthermore, the competing endogenous RNA mechanism between circROBO1 and BRCA1 was also explored, providing a reliable basis for the intrinsic regulatory mechanism of breast cancer.

Assessing the role of surface layer and molecular probe size in diffusion within meniscus tissue.

Diffusion within extracellular matrix is essential to deliver nutrients and larger metabolites to the avascular region of the meniscus. It is well known that both structure and composition of the meniscus vary across its regions; therefore, it is crucial to fully understand how the heterogenous meniscal architecture affects its diffusive properties. The objective of this study was to investigate the effect of meniscal region (core tissue, femoral, and tibial surface layers) and molecular weight on the diffusivity of several molecules in porcine meniscus. Tissue samples were harvested from the central area of porcine lateral menisci. Diffusivity of fluorescein (MW 332 Da) and three fluorescence-labeled dextrans (MW 3k, 40k, and 150k Da) was measured via fluorescence recovery after photobleaching. Diffusivity was affected by molecular size, decreasing as the Stokes' radius of the solute increased. There was no significant effect of meniscal region on diffusivity for fluorescein, 3k and 40k dextrans (p>0.05). However, region did significantly affect the diffusivity of 150k Dextran, with that in the tibial surface layer being larger than in the core region (p = 0.001). Our findings contribute novel knowledge concerning the transport properties of the meniscus fibrocartilage. This data can be used to advance the understanding of tissue pathophysiology and explore effective approaches for tissue restoration.

UIO66 low background signal and fluorescence synergism strategy for highly sensitive detection of Salmonella typhimurium.

Successful construction of a detection method for Salmonella typhimurium (S. typhimurium) based on the synergy of hybridization chain reaction (HCR) and fluorescence was realized in this paper. First, the aptamer modified with the quenching group Black Hole Quencher-1 acid (BHQ1) was immobilized on the magnetic beads in combination with the complementary chain of the aptamer modified with 6-carboxyfluorescein (6-FAM). Second, S. typhimurium and cDNA-6-FAM immobilized on magnetic beads competitively bound to the aptamer. Finally, the cDNA-6-FAM was released after magnetic separation acted as a promoter to trigger HCR amplification when the target presented. The fluorescence signal could be significantly improved by the combination of green SYBR Green I (SGI) and HCR long double-stranded DNA and the fluorescent synergy of 6-FAM and SGI. Because of the separation of target and its aptamer, the trigger strand was abstracted by magnetic separation. There was no HCR to generate long double-stranded DNA, and the fluorescence of excess hairpin/SGI could be adsorbed through UIO66 so that only a very low background signal was detected. This fluorescent sensor was capable of monitoring S. typhimurium in the range of 10-3.2 × 107 CFU mL-1 with a limit of detection as low as 1.5 CFU mL-1. Because of the excellent properties of the aptasensor and the validity of SGI fluorescence synergy, this HCR enzyme-free amplification strategy could be generalized to other areas.

Photoresponsive Azobenzene Nanocluster-Modified Liposomes: Mechanism Analysis Combining Experiments and Simulations.

Stimuli-responsive behaviors and controlled release in liposomes are pivotal in nanomedicine. To this end, we present an approach using a photoresponsive azobenzene nanocluster (AzDmpNC), prepared from azobenzene compounds through melting and aggregation. When integrated with liposomes, they form photoresponsive vesicles. The morphology and association with liposomes were investigated by using transmission electron microscopy. Liposomes loaded with calcein exhibited a 9.58% increased release after UV exposure. To gain insights into the underlying processes and elucidate the mechanisms involved. The molecular dynamic simulations based on the reactive force field and all-atom force field were employed to analyze the aggregation of isomers into nanoclusters and their impacts on phospholipid membranes, respectively. The results indicate that the nanoclusters primarily aggregate through π-π and T-stacking forces. The force density inside the cis-isomer of AzDmpNC formed after photoisomerization is lower, leading to its easier dispersion, rapid diffusion, and penetration into the membrane, disrupting the densification.

Far-red light-triggered cargo release from liposomes b ound to a photosensitizer-cellulose nanofiber hydrogel.

In our research we used the anionic nanofibrillar cellulose (ANFC) as a platform for far-red light-induced release of cargo from liposomes. In contrast to previous works, where photosensitizers are usually in the liposomal bilayers, we used a cellulose-binding dye. Our phthalocyanine derivative has been shown to bind very strongly to cellulose and cellulose nanofiber hydrogels, allowing us to place it outside of the liposomes. Both the sensitizer and cationic liposomes bind strongly to the ANFC after mixing, making the system easy to fabricate. Upon light activation, the photosensitizer generates reactive oxygen species (ROS) within the ANFC hydrogel, where the reactive oxygen species oxidize unsaturated lipids in the liposomal membrane, which makes the liposomes more permeable, resulting in on-demand cargo release. We were able to achieve ca. 70 % release of model hydrophilic cargo molecule calcein from the hydrogels with a relatively low dose of light (262 J/cm2) while employing the straightforward fabrication techniques. Our system was remarkably responsive to the far-red light (730 nm), enabling deep tissue penetration. Therefore, this very promising novel cellulose-immobilized photosensitizer liposomal platform could be used as a controlled drug delivery system, which can have applications in externally activated coatings or implants.

Discrepancies between transcutaneous and estimated glomerular filtration rates in rats with chronic kidney disease.

Accurate assessment of the glomerular filtration rate (GFR) is crucial for researching kidney disease in rats. Although validation of methods that assess GFR is crucial, large-scale comparisons between different methods are lacking. Both transcutaneous GFR (tGFR) and a newly developed estimated GFR (eGFR) equation by our group provide a low-invasive approach enabling repeated measurements. The tGFR is a single bolus method using FITC-labeled sinistrin to measure GFR based on half-life of the transcutaneous signal, whilst the eGFR is based on urinary sinistrin clearance. Here, we retrospectively compared tGFR, using both 1- and 3- compartment models (tGFR_1c and tGFR_3c, respectively) to the eGFR in a historic cohort of 43 healthy male rats and 84 male rats with various models of chronic kidney disease. The eGFR was on average considerably lower than tGFR-1c and tGFR-3c (mean differences 855 and 216 µL/min, respectively) and only 20 and 47% of measurements were within 30% of each other, respectively. The relative difference between eGFR and tGFR was highest in rats with the lowest GFR. Possible explanations for the divergence are problems inherent to tGFR, such as technical issues with signal measurement, description of the signal kinetics, and translation of half-life to tGFR, which depends on distribution volume. The unknown impact of isoflurane anesthesia used in determining mGFR remains a limiting factor. Thus, our study shows that there is a severe disagreement between GFR measured by tGFR and eGFR, stressing the need for more rigorous validation of the tGFR and possible adjustments to the underlying technique.

Quantifiable and Inexpensive Cell-Free Fluorescent Method to Confirm the Ability of Novel Compounds to Chelate Iron.

Cancer cells require large amounts of iron to maintain their proliferation. Iron metabolism is considered a hallmark of cancer, making iron a valid target for anti-cancer approaches. The development of novel compounds and the identification of leads for further modification requires that proof of mechanism assays be carried out. There are many assays to evaluate the impact on proliferation; however, the ability to chelate iron is an important and sometimes overlooked end-point measure due to the high costs of equipment and the challenge to quickly and reproducibly quantify the strength of chelation. Here, we describe a quantifiable and inexpensive cell-free fluorescent method to confirm the ability of novel compounds to chelate iron. Our assay relies on the commercially available inexpensive fluorescent dye Calcein, whose fluorescence can be quantified on most fluorescent microtiter plate readers. Calcein is a weak iron chelator, and its fluorescence is quenched when it binds Fe2+/3+; fluorescence is restored when a novel chelator outcompetes Calcein for bound Fe2+/3+. The removal of fluorescent quenching and the resulting increase in fluorescence allows the chelation ability of a novel putative chelator to be determined. Therefore, we offer an inexpensive, high-throughput assay that allows the rapid screening of novel candidate chelator compounds.

Melatonin improves the viability and ultrastructure of bovine oocyte-granulosa complexes of in vitro cultured early antral follicles.

This study aims to investigate the effects of melatonin on follicular growth, viability and ultrastructure, as well as on the levels of mRNA for antioxidant enzymes, reactive oxygen species (ROS) and meiotic progression in oocytes from in vitro cultured bovine early antral follicles. To this end, isolated early antral follicles (500-600 µm) were cultured in TCM-199+ alone or supplemented with 10-6 , 10-7 or 10-8 M melatonin at 38.5°C with 5% CO2 for 8 days. Follicle diameters were evaluated at days 0, 4 and 8 of culture. At the end of culture, ultrastructure, chromatin configuration, viability (calcein-AM and ethidium homodimer-1 staining), and the levels of ROS and mRNA for catalase (CAT), superoxide dismutase (SOD) and peroxiredoxin 6 (PRDX6) and glutathione peroxidase (GPx) were investigated in oocyte-granulosa cell complexes (OGCs). The results showed that early antral follicles cultured with 10-6 and 10-8 M melatonin had a progressive and significant increase in their diameters throughout the culture period (p < .05). Additionally, oocytes from follicles cultured with 10-7 or 10-8 M melatonin had increased fluorescence for calcein-AM, while those cultured with 10-6 or 10-7 M had reduced fluorescence for ethidium homodimer-1. Different from follicles cultured in other treatments, those cultured with 10-8 M melatonin had well-preserved ultrastructure of oocyte and granulosa cells. Melatonin, however, did not influence the levels of ROS, the mitochondrial activity, oocyte meiotic resumption and expression mRNA for SOD, CAT, GPX1 and PRDX6. In conclusion, the presence of 10-8 M melatonin in culture medium improves viability and preserves the ultrastructure of oocyte and granulosa cells of early antral follicles cultured in vitro.

Analysis and Development of Antigen-specific T Cells Derived from Peripheral Blood Mononuclear Cells of Healthy Donors.

BACKGROUND/AIM: Adoptive cell therapy using antigen-specific T cells is a promising treatment modality for cancer patients. Various methods to isolate specific T cells and identify corresponding T cell receptor (TCR) sequences are known. This study aimed to identify antigen-specific TCR from T cells isolated using carboxyfluorescein succinimidyl ester (CFSE), which marks proliferating activated T cells. MATERIALS AND METHODS: CFSE stained healthy donor peripheral blood mononuclear cells (PBMCs) were treated with cytomegalovirus (CMV) or Epstein-Barr virus (EBV) peptides for seven days. Then, proliferating T cells with decreased CFSE staining were isolated and single cell VDJ sequencing was performed on isolated T cells to identify antigen-specific TCRs. RESULTS: As antigen-specific TCR candidates, ten TCR clones were selected for the CMV antigen and five for the EBV antigen. The reactivity of ten CMV TCR-transduced T cells and one EBV TCR-transduced T cell toward T2 cells pulsed with CMV or EBV peptide was confirmed via NFAT-luciferase, IFN-γ ELISA, and cytotoxicity assays. CONCLUSION: Identification of antigen-specific TCRs with CFSE staining is a valid method for the development of effective immunotherapy. The identified CMV- or EBV-specific TCRs can be used for adoptive cell therapy to treat cancer.

Visualization of bone formation in sheep's middle ear by using fluorochrome sequential labelling (FSL).

One factor for the lacking integration of the middle ear stapes footplate prosthesis or the missing healing of stapes footplate fractures could be the known osteogenic inactivity. In contrast, it was recently demonstrated that titanium prostheses with an applied collagen matrix and immobilised growth factors stimulate osteoblastic activation and differentiation on the stapes footplate. Regarding those findings, the aim of this study was to evaluate the potential of bone regeneration including bone remodeling in the middle ear. Ten one-year-old female merino sheep underwent a middle ear surgery without implantation of middle ear prostheses or any other component for activating bone formation. Post-operatively, four fluorochromes (tetracycline, alizarin complexion, calcein green and xylenol orange) were administered by subcutaneous injection at different time points after surgery (1 day: tetracycline, 7 days: alizarin, 14 days: calcein, 28 days: xylenol). After 12 weeks, the temporal bones including the lateral skull base were extracted and histologically analyzed. Fluorescence microscopy analysis of the entire stapes with the oval niche, but in particular stapes footplate and the Crura stapedis revealed evidence of new bone formation. Calcein was detected in all and xylenol in 60% of the animals. In contrast, tetracycline and alizarin could only be verified in two animals. The authors were able to demonstrate the osseoregenerative potential of the middle ear, in particular of the stapes footplate, using fluorescence sequence labelling.

Malignance-restriction activity exhibited by bioactive compounds of selected actinobacteria as silver nanoparticles against A549 lung cancer cell lines.

This article deals with the antibacterial and anticancer potential of secondary metabolites produced by actinomycetes also reported as actinobacteria, Microbacterium proteolyticum (MN560041), and Streptomycetes rochei, where preliminary studies were done with the well diffusion method. These actinobacteria's silver nanoparticles were synthesized and characterized using transmission electron microscopy (TEM) and UV-Visible spectroscopy. Anticancer was measured using the MTT test, reactive oxygen species (ROS) generation measured with DCFDA, mitochondrial membrane potential (MMP) measurement, and DAPI fluorescence intensity activity was measured in treated and non-treated cancerous cells. The IC50 value for 5-FU (a), LA2(O) (b), LA2(R) (c), LA2(ON) (d), and LA2(RN) (e) was obtained at 3.91 µg/mL (52.73% cell viability), 56.12 µg/mL (52.35% cell viability), 44.90 µg/mL (52.3% cell viability), 3.45 µg/mL (50.25% cell viability), and 8.05 µg/mL (48.72% cell viability), respectively. TEM micrographs revealed discrete, well-separated AgNPs particles of size 7.88 ± 2 to 12.86 ± 0.24 nm. Gas chromatography-mass spectrometry was also performed to detect the compounds in bioactive metabolites where n-hexadecanoic acid was obtained as the most significant one. MTT test showed a substantial decline in A549 cell viability (up to 48.72%), 2.75-fold increase in ROS generation was noticed in comparison to untreated A549 lung cancer cells when measured with DCFDA. A total of 0.31-fold decrease in MMP and 1.74-fold increase in DAPI fluorescence intensity compared to untreated A549 lung cancer cells suggests that the synthesized nanoparticles promote apoptosis in cancerous cells. Our findings suggests that the secondary metabolites of M. proteolyticum and S. rochei in nanoparticle form can be used as a significant compound against lung cancers.

Study of cytotoxicity in neuroblastoma cell line exposed to patulin and citrinin.

Mycotoxins can be found in food and feed storage as well as in several kinds of foodstuff and are capable of harming mammals and some of them even in small doses. This study investigated on the undifferentiated neuronal cell line SH-SY5Y the effects of two mycotoxins: patulin (PAT) and citrinin (CTN), which are predominantly produced by fungi species Penicillium and Aspergillus. Here, the individual and combined cytotoxicity of PAT and CTN was investigated using the cytotoxic assay MTT. Our findings indicate that after 24 h of treatment, the IC50 value for PAT is 2.01 µM, which decreases at 1.5 µM after 48 h. In contrast, CTN did not attain an IC50 value at the tested concentration. Therefore, we found PAT to be the more toxic compared to CTN. However, the combined treatment suggests an additive toxic effect. With 2,7-dichlorodihydrofluorescin diacetate (DCFH-DA) DCFH-DA assay, ROS generation was demonstrated after CTN treatment, but PAT showed only small changes. The mixture presented a very constant behavior over time. Finally, the median-effect/combination index (CI-) isobologram equation demonstrated an additive effect after 24 h, but an antagonistic effect after 48 h for the interaction of the two mycotoxins.

Mitigation effect of hesperidin on X-ray radiation-induced intestinal barrier dysfunction in Caco-2 cell monolayers.

The tight junctions (TJs) and barrier function of the intestinal epithelium are highly sensitive to radiation. However, polyphenols can be used to reverse the effects of radiation. Here, we investigated the effects of hesperidin (hesperetin-7-rhamnoglucoside) on X-ray-induced intestinal barrier dysfunction in human epithelial Caco-2 monolayers. To examine whether hesperidin mitigated the effects of X-ray exposure (2 Gy), cell survival was evaluated and intestinal barrier function was assessed by measuring the transepithelial flux, apparent permeability coefficient (Papp), and barrier integrity. Hesperidin improved the survival of Caco-2 cell monolayers and attenuated X-ray exposure-induced intestinal barrier dysfunction. For fluorescein transport experiments, transepithelial flux and Papp of fluorescein in control group were significantly elevated by X-ray, but were restored to near control by 10 µM hesperidin pretreatment. Further, X-ray exposure decreased the barrier integrity and TJ interruption by reducing TJ-related proteins occludin and claudin-4, whereas cell monolayers pretreated with hesperidin before X-ray exposure were reinstated to control level. It was concluded that hesperidin treatment before X-ray exposure alleviated X-ray-induced intestinal barrier dysfunction through regulation of TJ-related proteins. These results indicate that hesperidin prevents and mitigates X-ray-induced intestinal barrier dysfunction.

Development of a closed-tube, calcein-based loop-mediated isothermal amplification assay to detect Salmonella spp. in raw meat samples.

Foodborne pathogens compromise food safety and public health, and Salmonella spp. are among the major pathogenic bacteria that cause outbreaks worldwide. Proper surveillance through timely and cost-effective detection methods across the food animal production chain is crucial to prevent Salmonella outbreaks and agricultural losses. Traditional culture methods are labor- and resource-intensive, with lengthy turnaround times. Meanwhile, conventional molecular tools, such as PCR and qPCR, are expensive and require technical skills and equipment. Loop-mediated isothermal amplification (LAMP) is a simple, rapid, inexpensive, highly sensitive, and specific molecular assay that does not require expensive equipment. Hence, this study developed and optimized a closed-tube, calcein-based LAMP assay to detect Salmonella using the invA gene and performed evaluation and validation against conventional PCR. The LAMP assay showed high specificity and sensitivity. It showed 10-fold higher sensitivity than conventional PCR, at <1 ng/µL DNA concentrations. Meanwhile, for CFU/mL, LAMP assay showed 1000-fold higher sensitivity than conventional PCR at 4.8 × 103 cells/mL than 4.8 × 107 cells/mL, respectively. For parallel testing of 341 raw meat samples, after conventional culture enrichment (until Rappaport-Vassiliadis broth), the optimized LAMP assay showed 100% detection on all samples while conventional PCR showed 100%, 99.04%, and 96.64% for raw chicken, beef, and pork samples, respectively. Meanwhile, a shortened enrichment protocol involving 3-h incubation in buffered peptone water only, showed lower accuracy in tandem with the optimized LAMP assay ranging from 55 to 75% positivity rates among samples. These suggest that the optimized LAMP assay possesses higher sensitivity over conventional PCR for invA gene detection when coupled with conventional enrichment culture methods. Hence, this assay has potential as a powerful complementary or alternative Salmonella detection method to increase surveillance capacity and protect consumer food safety and public health worldwide.

Mesoporous Silica Nanoparticles-Enhanced Microarray Technology for Highly Sensitive Simultaneous Detection of Multiplex Foodborne Pathogens.

Ensuring food safety is paramount for the food industry and global health concerns. In this study, we have developed a method for the detection of prevalent foodborne pathogenic bacteria, including Escherichia coli, Salmonella spp., Listeria spp., Shigella spp., Campylobacter spp., Clostridium spp., and Vibrio spp., utilizing antibody-aptamer arrays. To enhance the fluorescence signals on the microarray, the mesoporous silica nanoparticles (MSNs) conjugated with fluorescein, streptavidin, and seven detection antibodies-biotin were employed, forming fluorescein doped mesoporous silica nanoparticles conjugated with detection antibodies (MSNs-Flu-SA-Abs) complexes. The array pattern was designed for easy readability and enabled the simultaneous detection of all seven foodborne pathogens, referred to as the 7FP-biochip. Following the optimization of MSNs-Flu-SA-Abs complexes attachment and enhancement of the detection signal in fluorescent immunoassays, a high level of sensitivity was achieved. The detection limits for the seven pathogens in both buffer and food samples were 102 CFU/mL through visual screening, with fluorescent intensity quantification achieving levels as low as 20-34 CFU/g were achieved on the antibody-aptamer arrays. Our antibody-aptamer array offers several advantages, including significantly reduced nonspecific binding with no cross-reaction between bacteria. Importantly, our platform detection exhibited no cross-reactivity among the tested bacteria in this study. The multiplex detection of foodborne pathogens in canned tuna samples with spiked bacteria was successfully demonstrated in real food measurements. In conclusion, our study presents a promising method for detecting multiple foodborne pathogens simultaneously. With its high sensitivity and specificity, the developed antibody-aptamer array holds great potential for enhancing food safety and public health.

Detection of Nanoparticle-Mediated Total Oxidative Stress in T Cells Using CM-H2DCFDA Dye.

Oxidative stress is commonly observed in cells following exposure to nanoparticles. Both negative (e.g., cytotoxicity and inflammation) and beneficial (e.g., anti-inflammatory and tumor growth inhibiting) responses have been linked in the literature to oxidative stress, emphasizing the importance of developing methodologies to study this phenomenon in cells following their exposure to nanoparticles. In the protocol described herein, primary human T cells isolated from the peripheral blood of healthy donor volunteers are treated with nanoparticles and controls, and the generation of reactive oxygen species is detected by flow cytometry using CM-H2DCFDA reagent.

Evaluation of Nanomedicine Tissue Distribution and Stability by Alexa Fluor 488 and PEG Immunohistochemistry.

A primary issue with nanomedicine biological evaluation is determination of nanoparticle carrier tissue distribution and stability. Here we present a method to evaluate nanomedicine distribution in tissues that is applicable to most nanomedicine constructs. This method utilizes immunohistochemical (IHC) analysis of an Alexa Fluor 488-tag and/or polyethylene glycol (PEG), a very common nanomedicine component, for tissue localization. Using specific Alexa Fluor 488- and/or PEG antibody-based IHC staining procedures allows evaluation of high-resolution nanoparticle tissue distribution, nanoparticle tissue stability, and also allows correlation of distribution with morphological changes. This protocol outlines the methods to follow to ensure proper tissue collection and optimized immunohistochemical staining of Alexa Fluor 488-tag and PEG in tissues.

Human amniotic MSCs-mediated anti-inflammation of CD206hiIL-10hi macrophages alleviates isoproterenol-induced ventricular remodeling in mice.

BACKGROUND: Human amniotic mesenchymal stem cells (hAMSCs) derived from amniotic membrane have multilineage differentiation, immunosuppressive, and anti-inflammation which makes them suitable for the treatment of various diseases. OBJECTIVE: This study aimed to explore the therapeutic effect and molecular mechanism of hAMSCs in ventricular remodeling (VR). METHODS: hAMSCs were characterized by a series of experiments such as flow cytometric analysis, immunofluorescence, differentiative induction and tumorigenicity. Mouse VR model was induced by isoproterenol (ISO) peritoneally, and the therapeutic effects and the potential mechanisms of hAMSCs transplantation were evaluated by echocardiography, carboxy fluorescein diacetate succinimidyl ester (CFSE) labeled cell tracing, histochemistry, qRT-PCR and western blot analysis. The co-culturing experiments were carried out for further exploring the mechanisms of hAMSCs-derived conditioned medium (CM) on macrophage polarization and fibroblast fibrosis in vitro. RESULTS: hAMSCs transplantation significantly alleviated ISO-induced VR including cardiac hypertrophy and fibrosis with the improvements of cardiac functions. CFSE labeled hAMSCs kept an undifferentiated state in heart, indicating that hAMSCs-mediated the improvement of ISO-induced VR might be related to their paracrine effects. hAMSCs markedly inhibited ISO-induced inflammation and fibrosis, seen as the increase of M2 macrophage infiltration and the expressions of CD206 and IL-10, and the decreases of CD86, iNOS, COL3 and αSMA expressions in heart, suggesting that hAMSCs transplantation promoted the polarization of M2 macrophages and inhibited the polarization of M1 macrophages. Mechanically, hAMSCs-derived CM significantly increased the expressions of CD206, IL-10, Arg-1 and reduced the expressions of iNOS and IL-6 in RAW264.7 macrophages in vitro. Interestingly, RAW264.7-CM remarkably promoted the expressions of anti-inflammatory factors such as IL-10, IDO, and COX2 in hAMSCs. Furthermore, the CM derived from hAMSCs pretreated with RAW264.7-CM markedly inhibited the expressions of fibrogenesis genes such as αSMA and COL3 in 3T3 cells. CONCLUSION: Our results demonstrated that hAMSCs effectively alleviated ISO-induced cardiac hypertrophy and fibrosis, and improved the cardiac functions in mice, and the underlying mechanisms might be related to inhibiting the inflammation and fibrosis during the ventricular remodeling through promoting the polarization of CD206hiIL-10hi macrophages in heart tissues. Our study strongly suggested that by taking the advantages of the potent immunosuppressive and anti-inflammatory effects, hAMSCs may provide an alternative therapeutic approach for prevention and treatment of VR clinically.