In order to further evaluate ACE2 expression in pancreas sections, the same ACE2 IHC protocol was applied to other two primary antibodies anti-Human ACE2: monoclonal rabbit anti-Human ACE2 (cat. Figure S8 High Content Screening & Imaging Instruments | PerkinElmer. do Mar So Bernardo do Campo - SP motorcycle tuning tachometer mystic navy jordan 1 shirt | bridge card suppliers Finally, the filter was discarded, and the solution was desalted with OASIS cartridges according to the manufacturers instructions. The IDEAS software is available for free download through the Flow Core. Briefly, the pelleted cells were resuspended in QIAzol (cat. To identify ACE2, three different antibodies were used: #{"type":"entrez-nucleotide","attrs":{"text":"Ab108252","term_id":"41224589","term_text":"AB108252"}}Ab108252, #Ab15348 (Abcam), and #MAB933 (R&D system) were respectively diluted 1:1,000, 1:500, and 1:250 in 5% non-fat dry milk in TBST 1 and incubated o/n at +4C and then with Goat anti-rabbit (#111-036-003, Jackson Laboratories) or Goat anti-Mouse (#115-036-003, Jackson Laboratories) diluted 1:5,000 in 2% non-fat dry milk in TBST 1 1h RT. "Assay development guidelines for image-based high content screening, high content analysis and high content imaging." Fadista J, Vikman P, Laakso EO, Mollet IG, Esguerra JL, Taneera J, et al. The cells were subsequently washed with PBS without Ca2+ and Mg2+ and then incubated with monoclonal mouse anti-Human ACE2 (cat. Light transferring between neighbor pixels is in exact synchrony with the velocity of the cell stream to prevent blur and maintain a 0.5 um pixel resolution. Sardu C, DOnofrio N, Balestrieri ML, Barbieri M, Rizzo MR, Messina V, et al. mk7 golf bonnet release broken; lab made white sapphire rings; emerson and friends headbands. ). More and more researchers are turning to 3D cell models for high-content screening to better understand diseases and to test potential therapeutics. Dorrell C, Schug J, Lin CF, Canaday PS, Fox AJ, Smirnova O, et al. 4.3. South Miami Campus. Table S2 SARS-CoV-2 entry factors are highly expressed in nasal epithelial cells together with innate immune genes. A0564-Agilent Technologies, Santa Clara, CA, USA) diluted 1:1740 in BSA 1% in PBS without Ca2+ and Mg2+and with monoclonal mouse anti-Human ACE2 (cat. Subsequently, the sections were incubated with one drop of 3,3 Diaminobenzidine (DAB) chromogen solution (cat. In order to confirm the ACE2 cellular distribution observed in the pancreas using MAB933 antibody, we tested two additional anti-ACE2 antibodies from Abcam: Ab15348 and {"type":"entrez-nucleotide","attrs":{"text":"Ab108252","term_id":"41224589","term_text":"AB108252"}}Ab108252 (see Figure 7A Exposure of EndoC-H1 human beta-cell line and human pancreatic islets to pro-inflammatory cytokines significantly increased ACE2 expression. A human pluripotent stem cell-based platform to study SARS-CoV-2 tropism and model virus infection in human cells and organoids. Whole pancreata were processed following standardized procedures at the University of Pisa. The Opera Phenix Plus High-Content Screening System is the premier confocal solution for today's most demanding high content applications. Operetta CLS is a high throughput microplate imager for high-content analysis (HCA). Dotta F, Censini S, van Halteren AGS, Marselli L, Masini M, Dionisi S, et al. Interferons and viruses induce a novel truncated ACE2 isoform and not the full-length SARS-CoV-2 receptor. High content imaging aims to maximize data capture within imaging experiments. Results were expressed as mean SD. This makes specifics in HCI difficult to define, but in general, HCI involves performing normal imaging thousands or millions of times in order to maximize data capture effectively. FiguresS1B, C Coate KC, Cha J, Shrestha S, Wang W, Fasolino M, Morgan A, et al. Cellular imaging enables the detection and analysis of a myriad of cellular components from macromolecules to organelles and cell compartments. : The region of interest (ROI) was drawn to calculate the colocalization rate (which indicates the extent of colocalization between two different channels and reported as a percentage) as a ratio between the colocalization area and the image foreground. ). D8517, Sigma Aldrich, St. Louis, MO, USA) diluted 1:3,000 in PBS 1 and then mounted with Vectashield antifade medium (cat. Benefit from higher throughput, improved productivity, reduced variability, savings on reagent costs and more, by automating your Operetta CLS or Opera Phenix Plus system. Machine learning techniques enable supervised and unsupervised feature selection and clustering of highly multi-parametric data sets. Figure 3D (E) Mean intensity imaging analysis related to data reported in (C) of EndoC-H1 treated or not with cytokines. The central role played by ACE2 in SARS-CoV-2 infection has been further supported by evidence that SARS-CoV-2 infection is driven by ACE2 expression level (6). #16. High content imaging is primarily concerned with the automated analysis of large cell populations where the goal is to process as many cells as possible in the fastest time with the highest resolution. Our range of high-content analysis products includes: Imaging systems high content imaging perkin elmer. Field Applications Scientist, High Content Imaging PerkinElmer, Inc. Jan 2020 - Present 3 years 5 months. SCBD33ISV-Sigma Aldrich, St. Louis, MO, USA) to reduce non-specific reactions. ). PhenoLOGIC machine learning option enables you to teach the software to create optimized algorithms for your experiment, segmentation and cell classification. ), but the {"type":"entrez-nucleotide","attrs":{"text":"Ab108252","term_id":"41224589","term_text":"AB108252"}}Ab108252 antibody did not show any positivity within the islet parenchyma ( Furthermore, it should be taken into consideration that ACE2 expression may vary greatly among individuals due to genetic or environmental factors (59). Facilities. However, the analysis of ACE2 expression distribution in FFPE pancreas sections from a T1D longstanding donor (see Using both MAB933 and Ab15348 we observed ACE2 signal in pancreatic islets which suggests that ACE2 is expressed in endocrine cells. Inclusion in an NLM database does not imply endorsement of, or agreement with, It can also be equipped with an optional liquid handling module for fast live-cell assays. More accurately target your object of interest for significantly reduced acquisition and analysis times with intelligent acquisition technology for HCS. In details, palmitate and inflammatory stresses have been induced respectively by 2 mM of Sodium Palmitate (cat. P-value was calculated using MannWhitney U test (p < 0.05). With our custom-designed optics, including optical filters, objectives and zoom lenses, you have the ideal view for any application. Importantly, these observations were obtained both in the human -cell line EndoC-H1 and in human primary pancreatic islet cells, as shown by qRT-PCR, RNA-seq datasets and immunofluorescence. The mixture was charged on a filter 10K Pall, incubated 30min RT and centrifuged 13,800g 30min. With intelligent image acquisition technology, you can find 3D cell models at low resolution and high speed, and then image that region of interest at high-resolution, cutting down on acquisition and analysis times as well as data volume. mini plastic baseball bats. A lipid nanoparticle platform for mRNA delivery through repurposing of Angiotensin II mediates angiotensin converting enzyme type 2 internalization and degradation through an angiotensin II type I receptor-dependent mechanism, The insulin secretory granule as a signaling hub. Such staining pattern strongly suggests the presence of ACE2 in pancreatic vascular pericytes which are tightly associated with endothelial cells. IR002-Agilent Technologies, Santa Clara, CA, USA) as second and third primary antibodies for 1h at room temperature (RT). This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). | Write your own review. For 3D cell models, confocal imaging yields the highest sensitivity, best signal-to-noise ratios, and highest X, Y, and Z resolution compared to widefield imaging, without losing throughput. To get better results from your high-content imaging system, you need better imaging microplates. 945268 INNODIA HARVEST. As described above, we report that in human pancreatic islets, ACE2 is enriched in insulin-producing -cells. (2015), where a specific type of neuron (medium spiny neuron) was identified, characterized, and quantified with HCI of an entire mouse brain slice (Fig.1A). In our study, localization of ACE2 in pancreatic islet endocrine cells was observed using two out of three different antibodies tested. ", "Malvern particle size analyzer: Accurate, user-friendly, excellent support. PerkinElmer Inc. All Rights Reserved. Rua Samarit, 1117 - Jardim das Laranjeiras - So Paulo/SP - Brasil - CEP 02518-080 Fone. To determine the ACE2 protein expression pattern in human pancreatic tissue, we first performed a colorimetric immunohistochemistry analysis to detect ACE2 on formalin-fixed paraffin embedded (FFPE) pancreatic sections obtained from seven (n = 7) adult non-diabetic multiorgan donors collected by the INNODIA EUnPOD biobank ( ACE2 mRNA RT-Real-Time PCR analysis in EndoC-H1 treated or not (CTR) with Palmitate (2.0 mM) (A) or with cytokines (IL-1 + IFN + TNF) (Cyt. values are shown. : 11 3868.6210 In all cases analyzed, including different blocks of the same case, a similar expression pattern of ACE2 was observed even though a certain degree of variability in terms of ACE2 staining intensity within the islets was noted ( Hassan AO, Case JB, Winkler ES, Thackray LB, Kafai NM, Bailey AL, et al. ). SARS-CoV-2 Reverse Genetics Reveals a Variable Infection Gradient in the Respiratory Tract. The Moment is a true global shutter CMOS camera with an ultra-compact form factor, powered through USB 3.2 Gen 2. (Change), 1998 111-036-003, Jackson ImmunoResearch, Philadelphia, PA, USA), diluted 1:1000 in PBS 1 for 1h at room temperature (RT). See product, Software Gurgul-Convey E, Mehmeti I, Pltz T, Jrns A, Lenzen S. Sensitivity profile of the human EndoC-H1 beta cell line to proinflammatory cytokines. 7031 SW 62nd Ave. South Miami, 33143. Edfors F, Hober A, Linderbck K, Maddalo G, Azimi A, Sivertsson , et al. Wijnant SR, Jacobs M, Van Eeckhoutte HP, Lapauw B, Joos GF, Bracke KR, et al. Images were acquired using Leica TCS SP5 confocal laser scanning microscope system (Leica Microsystems, Wetzlar, Germany). ) (24, 25); the short-ACE2 misses part of the N-terminal region targeted by {"type":"entrez-nucleotide","attrs":{"text":"Ab108252","term_id":"41224589","term_text":"AB108252"}}Ab108252 antibody. Next, EndoC-H1 cells were rinsed with PBS without Ca2+ and Mg2+ and incubated with goat anti-mouse-488 1:500 in 1% BSA in PBS without Ca2+ and Mg2+ or with goat anti-rabbit-488 diluted 1:500 in 1% BSA in PBS without Ca2+ and Mg2+for 30min and with goat-anti rabbit Alexa-Fluor 594 conjugate (cat. Of note, the results obtained through WB analysis are in line with the immunofluorescence signal which revealed that {"type":"entrez-nucleotide","attrs":{"text":"Ab108252","term_id":"41224589","term_text":"AB108252"}}Ab108252 only stained a minor fraction of EndoC-H1 and the obtained signal was mainly found on the plasma membrane ( Human pancreatic sections analyzed in this study were obtained from pancreata of brain-dead adult non-diabetic multiorgan donors within the European Network for Pancreatic Organ Donors with Diabetes (EUnPOD), a project launched in the context of the INNODIA consortium (www.innodia.eu). An idea of the workflow involved in HCI can be seen in Fig.2. Bioz Stars score: 99/100, based on 40 PubMed citations. Importantly, the same expression pattern was observed also in human pancreatic islets exposed to the same cytokine mix, as demonstrated by a 2.4 and 5.1 fold-increase in ACE2 mRNA expression following IL-1 + IFN or IFN treatment respectively (p < 0.0001) ( High-Content Imaging (HCI) is typically defined as lower throughput automated microscopy (<100,000 samples or data points) compared to high-content screening. A11029-Molecular Probe, ThermoFisher Scientific, Waltham, MA, USA) diluted 1:500 in PBS 1, as secondary antibodies for 1h. Sections were counterstained with DAPI and then mounted as described above. The work is supported by the Innovative Medicines Initiative 2 (IMI2) Joint Undertaking under grant agreement No. The sequence of interest was retrieved using Export Data function by selecting 1,000 bp upstream 5 Flanking Sequence (GRCh38:X:15602149:15603148:-1) and downloaded in FASTA format. The presence of ACE2 in pancreatic pericytes is of sure interest. See product, Location Agilent 2100 Bioanalyzer technology with RNA Pico chips (cat. Interventional radiology can be used to treat a number of conditions like blood clots, gallbladder stones, kidney stones, andcancer. Careers, Unable to load your collection due to an error. Perform unbiased spontaneous phenotyping with intact, fixed, or live . Water immersion objectives are much better than air objectives at capturing light and providing high X, Y, Z resolution, so you can capture crisp cellular details and see further into 3D structures. Read more in our article Five Top Tips for a Successful High-Content Screening Assay with a 3D Cell Model System. ACE2 immunohistochemistry staining pattern in human pancreatic islets using MAB933, Ab15348, and {"type":"entrez-nucleotide","attrs":{"text":"Ab108252","term_id":"41224589","term_text":"AB108252"}}Ab108252 antibodies. - 2023 PerkinElmer Inc. All Rights Reserved. The chromatographic analysis was performed on a column Acquity UPLC Waters CSH C18 130 (1mm 100mm, 1.7 m, Waters) using a linear gradient and the eluents were 0.1% formic acid in water (phase A) and 0.1% formic acid in acetonitrile (phase B). These results are in contrast with the two recent preprint manuscripts (57, 58) which failed to observe ACE2 expression in pancreatic islet endocrine cells. Wang S, Guo F, Liu K, Wang H, Rao S, Yang P, et al. Additional host co-factors, such as transmembrane protease TMPRSS2, cathepsin B/L, and furin protease, have been shown to enhance efficiency of SARS-CoV-2 cell entry by processing the S-protein and eliciting membrane fusion and syncytia formation (6). Briefly, the cellular extracts were mixed with 12.5 l of EtOH (100%) and transferred to the purification column filter membrane. Before Figure 5C Against this background, we speculate that: (i) upon activation, ACE2 can be internalized through endosome/lysosome pathway (81); (ii) in -cells, ACE2 trafficking to cell membrane may be mediated by insulin granules; (iii) in -cells the short-ACE2 isoform could be mainly localized in cytoplasm, while long-ACE2 in the plasma membrane; (iii) ACE2 can be secreted and found in a soluble form or within exosomes (82, 83). For each case, a total of 711 islets/section were analyzed. The obtained libraries were submitted to a second quality control before sequencing on an Illumina HiSeq 2500. Operetta CLS is a high throughput microplate imager for high-content analysis (HCA). Spagnuolo I, Patti A, Sebastiani G, Nigi L, Dotta F. The case for virus-induced type 1 diabetes. Scharfmann R, Pechberty S, Hazhouz Y, von Blow M, Bricout-Neveu E, Grenier-Godard M, et al. A negative control with only secondary antibody incubation (no primary antibody control sample) was also included in order to exclude potential background artifacts generated by the secondary antibody or the enzymatic detection reaction ( This study involved: HCI is a good fit for imaging brain slices, which can often be extremely dense with cells and structural/functional relationships. (F) High content screening analysis of Corrected Median Spot intensity of ACE2 signal in EndoC-H1 treated or not with cytokines. Panels-c and -d: identification of ACE2 granular spots within segmented cytoplasm in panels-a and -b. Carty N, Berson N, Tillack K, Thiede C, Scholz D, Kottig K, Sedaghat Y, Gabrysiak C, Yohrling G, Kammer Hvd, Ebneth A, Mack V, Munoz-Sanjuan I and Kwak S (2015) Characterization of HTT Inclusion Size, Location, and Timing in the zQ175 Mouse Model of Huntingtons Disease: An In Vivo High-Content Imaging Study. Figure S5A, white arrow ). Springer Links book: High Content Screening. ). : 11 3868.6200 - Fax. EndoC-H1 was cultured at 37C with 5% CO2 in coated flask (coating medium composition: DMEM high-glucose cat. Please contact your local representative for further details. Fresh lung tissue was maintained in PBS1X on ice immediately after surgery until ready for the RNA extraction. G7513), Penicillin/Streptomycin 1% (cat. The key parameters in high-content imaging - speed, sensitivity (or intensity) and resolution - cannot be optimized independently as altering one of them influe From cellular screening and imaging applications to high-throughput screening and genomics-based applications, custom explorer G3 integrated workstations provi Out of focus images and inaccurate data collection are common issues caused by microplates that have not been specifically designed for the unique challenges of Revvity's PhenoVue suite of cellular imaging reagents including cell painting kits, organelle and cell compartment stains, fluorescent-labeled secondary anti Inductively Coupled Plasma Mass Spectrometry (ICP-MS), Inductively Coupled Plasma Optical Emission Spectroscopy (ICP-OES & ICP-AES), Atomic Spectroscopy Consumables & Accessories, Chromatography & Mass Spectrometry Software, Food Quality - Product Parts, Reagents and Consumables, Gas Chromatography Mass Spectrometry (GC/MS), Liquid Chromatography Mass Spectrometry (LC/MS), Molecular Spectroscopy Consumables & Accessories, Thermal Analysis Consumables & Accessories, Sample Prep and Automation for Food Testing, Environmental Surface Testing in Food Industry, Chemicals Quality Assurance & Quality Control, QA/QC Control of Raw Materials & Products, Pharmaceutical Raw Material Identification, Automated Nucleic Acid Quantitation and Normalization, Automated Small Scale Protein Purification, Plate Storage Solutions for Liquid Handling Automation, Reservoirs, Troughs and Other Liquid Handlers Consumables, Biochemical Prenatal & Preeclampsia Testing, Targeted Protein Degradation Assays and Reagents, Nimbus AI Accelerated Analysis Pilot Program, Microplates for Next Generation Sequencing, Consumables & Accessories for Life Sciences, Mass Spectrometry - Translational Research System, Cell and Gene Therapy Research and Development Solutions, Automated Nucleic Acid Extraction Solutions, Buchser, William, et al. South Miami, 33143. Interventional radiology is theminimally invasive treatment of conditions like cancer, using imaging tools like CT Scans, MRI's, and Ultrasounds. This concept is supported by reports in which impaired glycemic control is associated with increased risk of severe COVID-19. Originally developed as a complementary technology to traditional biochemical high-throughput screening (HTS) in drug discovery, today high-content screening is established in a far broader area of the life science space as an unbiased imaging method to assess cellular function. Analysis of ACE2 mRNA expression in these cells demonstrated a similar expression level in comparison to human pancreatic islets ( Blodgett DM, Nowosielska A, Afik S, Pechhold S, Cura AJ, Kennedy NJ, et al. Overall n = 50 microdissected pancreatic islets from each case were analyzed. 4488593, ThermoFisher Scientific, Waltham, MA, USA) following manufacturers instructions. ). T8158) and Sodium selenite 6.7 ng/ml (cat. 1 offer from $388.88. Ramos-Rodrguez M, Raurell-Vila H, Colli ML, Alvelos MI, Subirana-Grans M, Juan-Mateu J, et al. Automated image analysis was performed using Harmony High-Content Imaging (PerkinElmer, Waltham, MA, USA), and fluorescence intensity of treated or untreated cells was measured based on Alexa-555 (insulin) and Alexa-488 (ACE2) fluorochromes. Activation of ACE2/angiotensin (1-7) attenuates pancreatic cell dedifferentiation in a high-fat-diet mouse model. Guan W-J, Liang W-H, Zhao Y, Liang H-R, Chen Z-S, Li Y-M, et al. The instrument manual is probably one of the easiest to follow manuals I've ever seen, it is informative and contains plenty of useful tips. Please note that product labeling (such as kit insert, product label, and kit box) may be different compared to the company branding. Integrates with robotic automation platforms for a fully automated solution. Figure S11 Of note, two recent preprint manuscripts also indicated the presence of ACE2 expression in pancreatic microvasculature (57, 58). See the other articles in our High Content Imaging sections to learn about hardware, analysis programs, and suitable cameras for this format of imaging. Scale bars in (BD) are 100 m. P-value was calculated using MannWhitney U test. Scale bars in (A, C, E) 150 m. Proteins were then transferred to Nitrocellulose 0.2 m membrane using wet electrophoresis system. Microscopic images for scientific research. Figure 5A Bramswig NC, Everett LJ, Schug J, Dorrell C, Liu C, Luo Y, et al. Operetta High Content Imaging System by PerkinElmer, Inc. By proceeding navigation on this page, you agree to the use of cookies according to the terms of our, Slicing a mouse brain and culturing the slices in neural growth medium, Staining each slice with multiple fluorescent markers for specific proteins, Acquiring hundreds of images at different XY positions at 40x magnification, Stitching these images together to get an overall image of an entire brain slice, Performing quantitative image analysis on specific fields with specialized software. But not totally inevitableoften only part of the total well area, where the 3D model is situated, is of interest. Dynamic regulation of SARS-Cov-2 binding and cell entry mechanisms in remodeled human ventricular myocardium. Scale bar in panels-d and -k: 100m. This in-situ ACE2 expression pattern data are in line with three different bulk RNA-seq datasets analyzing human - and -cells transcriptome, reporting higher expression of ACE2 mRNA in -cells as compared to -cells (6365) ( X0943-Agilent Technologies, Santa Clara, CA, USA) or negative isotype control rabbit IgG (cat. Taken together, our data indicate a potential link between SARS-CoV-2 and diabetes through putative infection of pancreatic microvasculature and/or ductal cells and/or through direct -cell virus tropism. Signals Image Artist ACE2 staining pattern in human pancreas. 7031 SW 62nd Ave. South Miami, 33143. Copyright 2023 Teledyne Photometrics. Alongside drug discovery, some of the main uses of HCI are studying cell behavior, mechanisms/molecular function, and safety/toxicology studies. Tel: 305-558-2500 | View, 7000 SW 62nd Ave, Suite 600. Indiana Biosciences Research Institute, Indianapolis, IN, United States, 7 Briefly, after deparaffinization and rehydration through decreasing alcohol series (see above), pancreatic sections were subjected to heat induced antigen retrieval using 10 mM citrate buffer pH 6.0 in microwave (600 W) for 10min. Software is very easy to use and user friendly. ACE2 mRNA expression in LCM-human pancreatic islets showed a consistent expression among cases, similar to isolated islets, as shown by ACE2 mRNA raw Ct and normalized values ( 0001) ( Mossel EC, Huang C, Narayanan K, Makino S, Tesh RB, Peters CJ. (A) Aligned sequences and structures of recently described ACE2 isoforms, long-ACE2 (805aa, ~110 kDa) and short-ACE2 (459aa, ~50kDa). Teuwen L-A, Geldhof V, Pasut A, Carmeliet P. Varga Z, Flammer AJ, Steiger P, Haberecker M, Andermatt R, Zinkernagel AS, et al. Surprisingly, we were not able to observe ACE2 pancreatic islets positive signal using Abcam monoclonal antibody {"type":"entrez-nucleotide","attrs":{"text":"Ab108252","term_id":"41224589","term_text":"AB108252"}}Ab108252, while signal was clearly evident in microvasculature and scattered ductal cells.
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