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Inflammasomes

Inflammasomes are multiprotein complexes that play a crucial role in the innate immune response.
These cytoplasmic sensors detect pathogen-associated molecular patterns and danger signals, triggering the activation of caspase-1 and the subsequent release of proinflammatory cytokines.
Inflammasomes are involved in the pathogenesis of various inflammatory diseases, including autoimmune disorders, neurodegenerative conditions, and metabolic syndromes.
Understanding the mechanisms and regulation of inflammasome activation is essential for developing targeted therapies to modulate inflammatory processes.
Researchers can leverage PubCompare.ai's AI-powered platform to effortlessly locate the best protocols from literature, preprints, and patents, and identify the most reproducible and accruate insights for their inflammasome research, boosting efficiency and unlocking new discoveries.

Most cited protocols related to «Inflammasomes»

1
Inflammasome Activation in Macrophages
Cited 29 times
BMDM were seeded at 5 ×0 105/ml or 1 × 106/ml, HMDM at 5 × 105/ml and PBMC at 2 × 106/ml or 5 ×0 106/ml in 96 well plates. The following day the overnight medium was replaced and cells were stimulated with 10 ng/ml LPS from Escherichia coli serotype EH100 (ra) TLRgrade™ (Alexis Biochemicals) for 3 h. Medium was removed and replaced with serum free medium (SFM) containing DMSO (1:1,000), MCC950 (0.001–10 µM), glyburide (200 µM) (Sigma Aldrich), parthenolide (10 µM) (Enzo Life Sciences) or Bayer cysteinyl leukotriene receptor antagonist 1-(5-carboxy-2{3-[4-(3-cyclohexylpropoxy)phenyl]propoxy}benzoyl)piperidine-4-carboxylic acid (40 µM) (Amgen Inc., Thousand Oaks, CA, USA) for 30 min. Cells were then stimulated with inflammasome activators: 5 mM adenosine 5’-triphosphate disodium salt hydrate (ATP) (1 h), 1 µg/ml Poly(deoxyadenylic-thymidylic) acid sodium salt (Poly dA:dT) (Sigma Aldrich) transfected with Lipofectamine 2000™ (Invitrogen) (3–4 h), 200 µg/ml MSU (overnight) and 10 µM nigericin (Invivogen) (1 h) or S. typhimurium UK-1 strain (M.O.I. 20) obtained from Dr. Sinead Corr, Trinity College Dublin, Ireland (2 h). Cells were also stimulated with 25 µg/ml Polyadenylic-polyuridylic acid (Invivogen) (4 h). For non-canonical inflammasome activation cells were primed with 100 ng/ml Pam3CSK4 (Invivogen) for 4 h, medium was removed and replaced with SFM containing DMSO or MCC950 and 2 µg/ml LPS was transfected using 0.25% FuGENE® (Promega) for 16 h. Supernatants were removed and analysed using ELISA kits according to the manufacturer’s instructions (DuoSet® R&D Systems or ReadySetGo!® eBioscience). LDH release was measured using the CytoTox96® non-radioactive cytotoxicity assay (Promega)
Coll R.C., Robertson A.A., Chae J.J., Higgins S.C., Muñoz-Planillo R., Inserra M.C., Vetter I., Dungan L.S., Monks B.G., Stutz A., Croker D.E., Butler M.S., Haneklaus M., Sutton C.E., Núñez G., Latz E., Kastner D.L., Mills K.H., Masters S.L., Schroder K., Cooper M.A, & O’Neill L.A. (2015). A small molecule inhibitior of the NLRP3 inflammasome is a potential therapeutic for inflammatory diseases. Nature medicine, 21(3), 248-255.
Publication 2015
Adenosine Triphosphate Biological Assay Carboxylic Acids Cells cysteinyl leukotriene receptor Cytotoxin Enzyme-Linked Immunosorbent Assay Escherichia coli FuGene Glyburide Inflammasomes Leukotriene Antagonists lipofectamine 2000 MCC-950 Nigericin parthenolide piperidine poly(dA) Poly A Poly A-U Promega Quercus Radioactivity Serum Sodium Sodium Chloride Strains Sulfoxide, Dimethyl Thymidine Monophosphate
2
Caspase-1 Regulates Alzheimer's Pathology
Cited 27 times
Caspase-1 activation of human and mouse brain tissue were analyzed by Western blot of cleaved caspase-1. IL-1β was quantified by ELISA. Microglial ASC speck formation was detected by immunohistochemistry. All mice were on C57/Bl6 background, including WT, NLRP3−/−,27 (link), APP/PS15 (link), APP/PS1/NLRP3−/−, Caspase-1−/−,28 (link), APP/PS1/Caspase-1−/− and were analyzed for cognitive function using the Morris Water Maze, the object recognition test and open field behavioural testing. Synaptic plasticity was determined by measuring long term potentiation (LTP) in acutely isolated hippocampal slices. Spine density was assessed by analyzing mid apical dendritic sections of pyramidal CA1 neurons. Cerebral Aβ load was determined by thioflavin-S-histochemistry of serial sections. Sequential extraction of homogenized brains by radio-immunoprecipitation assay, sodium dodecyl sulfate buffer and formic acid was employed to determine Aβ levels. Aβ nitration was determined by ELISA and immunohistochemistry using a specific antibodies against 3NTyr10 (link)-Aβ25 (link). Western blot detection was used to analyze the protein levels of APP, CTFs, Aβ, BACE1, IDE and NOS2. Inflammasome activation was confirmed by detection of ASC speck formation in microglia isolated from adult mouse. Microglial Aβ phagocytosis was determined after peripheral injection of methoxy-XO4, isolation of microglia and subsequent FACS analysis. Confirmatory immunocytochemistry was performed using antibody IC16 and the lysosomal marker LAMP2. Plaque morphology and microglial Aβ uptake was analyzed by coimmunostaining with Iba-1, methoxy-XO4 and IC16. mRNA levels of IDE, NEP, M1 and M2 markers were determined either from sorted microglia or from brain tissue by qPCR.
Heneka M.T., Kummer M.P., Stutz A., Delekate A., Schwartz S., Saecker A., Griep A., Axt D., Remus A., Tzeng T.C., Gelpi E., Halle A., Korte M., Latz E, & Golenbock D. (2012). NLRP3 is activated in Alzheimer´s disease and contributes to pathology in APP/PS1 mice. Nature, 493(7434), 674-678.
Publication 2012
Adult Antibodies BACE1 protein, human Biological Assay Brain Buffers Caspase 1 Cognition Dendrites Enzyme-Linked Immunosorbent Assay formic acid Histocytochemistry Homo sapiens Immunocytochemistry Immunoglobulins Immunohistochemistry Immunoprecipitation Inflammasomes Interleukin-1 beta isolation LAMP2 protein, human Long-Term Potentiation Lysosomes Mice, Laboratory Microglia Morris Water Maze Test Neuronal Plasticity Nitrates Nitric Oxide Synthase Type II Phagocytosis Proteins Pyramidal Cells RNA, Messenger Senile Plaques Sulfate, Sodium Dodecyl thioflavine Tissues Vertebral Column Western Blotting
3
Systematic Review of Inflammasome and Pyroptosis in Brain
Cited 16 times
We retrieved all literature data regarding the inflammasome/pyroptosis in brain indexed in the Web of Science (WOS) Core Collection (Clarivate Analysis, Boston, United States; http://apps.webofknowledge.com/WOS_GeneralSearch_input.do?product=WOS&SID=7DLaapxWMwSkqZf4LRr&search_mode=GeneralSearch). The term pyroptosis was detected with MeSH (https://www.ncbi.nlm.nih.gov/mesh), whereas the “pyroptosis”, “inflammasome”, and “brain” show other expressions, such as “pyroptotic” and “pyroptosome”. The articles from 2000 to 2020 (October 16, 2020) were searched, the language type was set to English, and the document type was set to article and review.
The search terms and strategies used for the WOS database are as follows: # 1, “pyroptosis”; # 2, “pyroptotic”; # 3, “inflammasome”; # 4, “pyroptosome”; # 5 “brain”; # 6, “# 1” OR “# 2” OR “# 3” OR “# 4”; # 7, “# 5” AND “# 6”.
A total of 14,343 documents of “inflammasome OR pyroptosis” and 1,222 documents of “inflammasome OR pyroptosis AND brain” were retrieved from WOS Core Collection, and then the documents were used to make visual analysis ultimately. The deadline for researched publications was October 16, 2020.
Chen Y., Li Y., Guo L., Hong J., Zhao W., Hu X., Chang C., Liu W, & Xiong K. (2021). Bibliometric Analysis of the Inflammasome and Pyroptosis in Brain. Frontiers in Pharmacology, 11, 626502.
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Publication 2020
Brain CTSB protein, human Inflammasomes Pyroptosis
4
Inflammasome Activation Analysis by Flow Cytometry
Cited 16 times
Sester and colleagues performed an FCM method to detect ASC redistribution in myeloid cell defining speck formation by changes in fluorescence peak height and width [2156 (link), 2157 (link)]. This protocol permits to define the true activated inflammasome by assessment ASC speck formation, because until then the detection of inflammasome, activation was made by monitor its end products, IL-1β or IL-18, or activated caspase-1 detection. Notably different pathways can secrete IL-1β and the methods to analyze caspase-1 activity are not always specific, in both cases the outcomes could be inflammasome independent. Despite to ASC speck formation microscopy analysis, used in the past, this methodology is faster, more accurate, and sensitive. Recently a better method to analyze simultaneously ASC speck and caspase-1 activity was performed by Amnis ImageStreamX [2158 (link)]. This protocol eliminates false positive events detected by flow-cytometry method, by specific masks to select only single cell, excluding cells with nonspecific-like aggregation of ASC and also defining ASC speck size. Finally the study analyze the presence and distribution of active caspase-1, detected by FLICA spots, and ASC speck simultaneously, evaluating inflammasome activation.
In this section we show the analysis of inflammasome activation by FlowSight, performed utilizing ASC speck formation in LPS+Nig stimulated-THP1derived macrophage.
Cossarizza A., Chang H.D., Radbruch A., Acs A., Adam D., Adam-Klages S., Agace W.W., Aghaeepour N., Akdis M., Allez M., Almeida L.N., Alvisi G., Anderson G., Andrä I., Annunziato F., Anselmo A., Bacher P., Baldari C.T., Bari S., Barnaba V., Barros-Martins J., Battistini L., Bauer W., Baumgart S., Baumgarth N., Baumjohann D., Baying B., Bebawy M., Becher B., Beisker W., Benes V., Beyaert R., Blanco A., Boardman D.A., Bogdan C., Borger J.G., Borsellino G., Boulais P.E., Bradford J.A., Brenner D., Brinkman R.R., Brooks A.E., Busch D.H., Büscher M., Bushnell T.P., Calzetti F., Cameron G., Cammarata I., Cao X., Cardell S.L., Casola S., Cassatella M.A., Cavani A., Celada A., Chatenoud L., Chattopadhyay P.K., Chow S., Christakou E., Čičin-Šain L., Clerici M., Colombo F.S., Cook L., Cooke A., Cooper A.M., Corbett A.J., Cosma A., Cosmi L., Coulie P.G., Cumano A., Cvetkovic L., Dang V.D., Dang-Heine C., Davey M.S., Davies D., De Biasi S., Del Zotto G., Cruz G.V., Delacher M., Bella S.D., Dellabona P., Deniz G., Dessing M., Di Santo J.P., Diefenbach A., Dieli F., Dolf A., Dörner T., Dress R.J., Dudziak D., Dustin M., Dutertre C.A., Ebner F., Eckle S.B., Edinger M., Eede P., Ehrhardt G.R., Eich M., Engel P., Engelhardt B., Erdei A., Esser C., Everts B., Evrard M., Falk C.S., Fehniger T.A., Felipo-Benavent M., Ferry H., Feuerer M., Filby A., Filkor K., Fillatreau S., Follo M., Förster I., Foster J., Foulds G.A., Frehse B., Frenette P.S., Frischbutter S., Fritzsche W., Galbraith D.W., Gangaev A., Garbi N., Gaudilliere B., Gazzinelli R.T., Geginat J., Gerner W., Gherardin N.A., Ghoreschi K., Gibellini L., Ginhoux F., Goda K., Godfrey D.I., Goettlinger C., González-Navajas J.M., Goodyear C.S., Gori A., Grogan J.L., Grummitt D., Grützkau A., Haftmann C., Hahn J., Hammad H., Hämmerling G., Hansmann L., Hansson G., Harpur C.M., Hartmann S., Hauser A., Hauser A.E., Haviland D.L., Hedley D., Hernández D.C., Herrera G., Herrmann M., Hess C., Höfer T., Hoffmann P., Hogquist K., Holland T., Höllt T., Holmdahl R., Hombrink P., Houston J.P., Hoyer B.F., Huang B., Huang F.P., Huber J.E., Huehn J., Hundemer M., Hunter C.A., Hwang W.Y., Iannone A., Ingelfinger F., Ivison S.M., Jäck H.M., Jani P.K., Jávega B., Jonjic S., Kaiser T., Kalina T., Kamradt T., Kaufmann S.H., Keller B., Ketelaars S.L., Khalilnezhad A., Khan S., Kisielow J., Klenerman P., Knopf J., Koay H.F., Kobow K., Kolls J.K., Kong W.T., Kopf M., Korn T., Kriegsmann K., Kristyanto H., Kroneis T., Krueger A., Kühne J., Kukat C., Kunkel D., Kunze-Schumacher H., Kurosaki T., Kurts C., Kvistborg P., Kwok I., Landry J., Lantz O., Lanuti P., LaRosa F., Lehuen A., LeibundGut-Landmann S., Leipold M.D., Leung L.Y., Levings M.K., Lino A.C., Liotta F., Litwin V., Liu Y., Ljunggren H.G., Lohoff M., Lombardi G., Lopez L., López-Botet M., Lovett-Racke A.E., Lubberts E., Luche H., Ludewig B., Lugli E., Lunemann S., Maecker H.T., Maggi L., Maguire O., Mair F., Mair K.H., Mantovani A., Manz R.A., Marshall A.J., Martínez-Romero A., Martrus G., Marventano I., Maslinski W., Matarese G., Mattioli A.V., Maueröder C., Mazzoni A., McCluskey J., McGrath M., McGuire H.M., McInnes I.B., Mei H.E., Melchers F., Melzer S., Mielenz D., Miller S.D., Mills K.H., Minderman H., Mjösberg J., Moore J., Moran B., Moretta L., Mosmann T.R., Müller S., Multhoff G., Muñoz L.E., Münz C., Nakayama T., Nasi M., Neumann K., Ng L.G., Niedobitek A., Nourshargh S., Núñez G., O’Connor J.E., Ochel A., Oja A., Ordonez D., Orfao A., Orlowski-Oliver E., Ouyang W., Oxenius A., Palankar R., Panse I., Pattanapanyasat K., Paulsen M., Pavlinic D., Penter L., Peterson P., Peth C., Petriz J., Piancone F., Pickl W.F., Piconese S., Pinti M., Pockley A.G., Podolska M.J., Poon Z., Pracht K., Prinz I., Pucillo C.E., Quataert S.A., Quatrini L., Quinn K.M., Radbruch H., Radstake T.R., Rahmig S., Rahn H.P., Rajwa B., Ravichandran G., Raz Y., Rebhahn J.A., Recktenwald D., Reimer D., e Sousa C.R., Remmerswaal E.B., Richter L., Rico L.G., Riddell A., Rieger A.M., Robinson J.P., Romagnani C., Rubartelli A., Ruland J., Saalmüller A., Saeys Y., Saito T., Sakaguchi S., de-Oyanguren F.S., Samstag Y., Sanderson S., Sandrock I., Santoni A., Sanz R.B., Saresella M., Sautes-Fridman C., Sawitzki B., Schadt L., Scheffold A., Scherer H.U., Schiemann M., Schildberg F.A., Schimisky E., Schlitzer A., Schlosser J., Schmid S., Schmitt S., Schober K., Schraivogel D., Schuh W., Schüler T., Schulte R., Schulz A.R., Schulz S.R., Scottá C., Scott-Algara D., Sester D.P., Shankey T.V., Silva-Santos B., Simon A.K., Sitnik K.M., Sozzani S., Speiser D.E., Spidlen J., Stahlberg A., Stall A.M., Stanley N., Stark R., Stehle C., Steinmetz T., Stockinger H., Takahama Y., Takeda K., Tan L., Tárnok A., Tiegs G., Toldi G., Tornack J., Traggiai E., Trebak M., Tree T.I., Trotter J., Trowsdale J., Tsoumakidou M., Ulrich H., Urbanczyk S., van de Veen W., van den Broek M., van der Pol E., Van Gassen S., Van Isterdael G., van Lier R.A., Veldhoen M., Vento-Asturias S., Vieira P., Voehringer D., Volk H.D., von Borstel A., von Volkmann K., Waisman A., Walker R.V., Wallace P.K., Wang S.A., Wang X.M., Ward M.D., Ward-Hartstonge K.A., Warnatz K., Warnes G., Warth S., Waskow C., Watson J.V., Watzl C., Wegener L., Weisenburger T., Wiedemann A., Wienands J., Wilharm A., Wilkinson R.J., Willimsky G., Wing J.B., Winkelmann R., Winkler T.H., Wirz O.F., Wong A., Wurst P., Yang J.H., Yang J., Yazdanbakhsh M., Yu L., Yue A., Zhang H., Zhao Y., Ziegler S.M., Zielinski C., Zimmermann J, & Zychlinsky A. (2019). Guidelines for the use of flow cytometry and cell sorting in immunological studies (second edition). European journal of immunology, 49(10), 1457-1973.
Publication 2019
Activation Analysis Caspase 1 Cells Exanthema Flow Cytometry Fluorescence Inflammasomes Interleukin-1 beta interleukin 18 protein, human Macrophage Microscopy Myeloid Progenitor Cells
5
Transcriptome Analysis of Inflammatory Mediators
Cited 12 times
An experimental data mining strategy was adopted to analyze the expression profiles of mRNA transcripts of TLRs, NLRs, inflammasome components, inflammatory caspases, proinflammatory cytokines IL-1β and IL-18 in vasculature-related tissues by mining human expression sequence tag (EST) database (Fig. 1)(19 (link)), which resulted from cDNA cloning from various tissue cDNA libraries and DNA sequencing followed by sequence analysese and deposited in the National Institutes of Health (NIH)/National Center of Biotechnology Information (NCBI) Unigene (http://www.ncbi.nlm.nih.gov/sites/entrez?db=unigene)(20 (link)). The arbitrary units of the gene expression were calculated by normalizing the transcripts per million of gene of interest with that of house-keeping gene β-actin in any given tissue. The confidential intervals of the expression variation of house-keeping genes were generated by calculating the mean and 2 times the standard deviation of the arbitrary units of three randomly selected house-keeping genes normalized by β-actin in given tissues (legend of Fig. 22). If the expression variation of a given gene in various tissues was larger than the upper limit of the confidential intervals (the mean plus 2 times the standard deviations) of the house-keeping genes, the high expression levels of genes in the tissues were statistically significant. Any given gene transcript, if lower than one per million, was technically presented as no expression.
Yin Y., Yan Y., Jiang X., Mai J., Chen N.C., Wang H, & Yang X.F. (2009). INFLAMMASOMES ARE DIFFERENTIALLY EXPRESSED IN CARDIOVASCUALR AND OTHER TISSUES. International journal of immunopathology and pharmacology, 22(2), 311-322.
Publication 2009
Actins Caspase cDNA Library Cytokine DNA, Complementary Gene Expression Genes Genes, Housekeeping Genetic Diversity Homo sapiens Inflammasomes Inflammation Interleukin-1 beta interleukin 18 protein, human RNA, Messenger Tissues

Most recents protocols related to «Inflammasomes»

1
Neuronal and Glial Cell Culture and Inflammasome Activation
SH-SY5Y human neuroblastoma cell line (ECACC, 94030304) and the mouse spontaneously immortalized microglia-9 (SIM-A9) cell line (ATCC-CRL-3265) [30 (link)] were cultured in Gibco™ DMEM/ F-12 with GlutaMAX™ (ThermoFisher, Waltham, MA, 31331028), supplemented with 10% fetal bovine serum (ThermoFisher, Waltham, MA, 10100139) and 5 μg/ml plasmocin™ (InvivoGen, San Diego, CA, ant-mpt). The culture medium of SIM-A9 cells was supplemented with 5% horse serum (Sigma-Aldrich, Saint Louis, MO, H1270). Embryonic cortical-hippocampal neurons from wild-type (WT) and SREBF-2 mice (B6;SJL-Tg(rPEPCKSREBF2)788Reh/J, RRID:IMSR_JAX:003311) were isolated on day 16–17 of pregnancy by trypsin digestion following a standard protocol [31 (link)]. Dissociated cells were grown in Neurobasal™ medium (ThermoFisher, 21103–049) supplemented with 2.5% (v/v) B27 supplement (ThermoFisher, 17504–001), 0.5 mM L-glutamine (Sigma-Aldrich, G7513) and 5 μg/ml plasmocinTM (InvivoGen, ant-mpt), and plated onto poly-D-lysine (Sigma-Aldrich, P6407)- and laminin (Sigma-Aldrich, L2020)-coated plates at a density of 2 × 105 cells/cm2. Half of the culture medium was changed every 3 or 4 days. Over 95% of neuronal purity was confirmed by immunochemistry using antibodies targeting neuronal and glial markers. Experiments were performed at 7 to 10 days in vitro (DIV). All procedures involving animals and their care were approved by the ethics committee of the University of Barcelona and were conducted in accordance with institutional guidelines in compliance with national and international laws and policies.
Cell cholesterol enrichment was achieved by incubation with a cholesterol:methyl-β-cyclodextrin complex (CHO:MCD; containing 50 μg/ml of cholesterol) (Sigma-Aldrich, C4951) for 1 h followed by 4-h recovery. To induce inflammasome activation, cells were treated with 10 μg/ml lipopolysaccharide (LPS) from Escherichia coli O111:B4 (Sigma-Aldrich, L4391), 10 μg/ml N-acetylmuramyl-L-alanyl-D-isoglutamine hydrate (also known as muramyl dipeptide, MDP; Sigma-Aldrich, A9519), 5 mM ATP (Sigma-Aldrich, A2383), 150 μg/ml monosodium urate crystals (MSU; Santa Cruz Biotech., sc-202711), and oligomeric Aβ at the indicated times. Preincubation with 4 mM glutathione ethyl ester (GSHee) or with the cell-permeable caspase 1 inhibitor I (10 μM; Bachem, 4095744) was performed 30 min before treatment when indicated.
de Dios C., Abadin X., Roca-Agujetas V., Jimenez-Martinez M., Morales A., Trullas R., Mari M, & Colell A. (2023). Inflammasome activation under high cholesterol load triggers a protective microglial phenotype while promoting neuronal pyroptosis. Translational Neurodegeneration, 12, 10.
Full text: Click here
Publication 2023
Acetylmuramyl-Alanyl-Isoglutamine Animals Antibodies Cell Lines Cells Cholesterol Cortex, Cerebral Culture Media Cyclodextrins Dietary Supplements Digestion Embryo Equus caballus Escherichia coli Ethics Committees Fetal Bovine Serum Glutamine Homo sapiens Inflammasomes interleukin-1beta-converting enzyme inhibitor isoglutamine Laminin Lipopolysaccharides Lysine Microglia Mus Neuroblastoma Neuroglia Neurons Permeability plasmocin Poly A Pregnancy S-ethyl glutathione Serum Sodium Urate Monohydrate Trypsin
2
Neuronal and Glial Cell Culture and Inflammasome Activation
SH-SY5Y human neuroblastoma cell line (ECACC, 94030304) and the mouse spontaneously immortalized microglia-9 (SIM-A9) cell line (ATCC-CRL-3265) [30 (link)] were cultured in Gibco™ DMEM/ F-12 with GlutaMAX™ (ThermoFisher, Waltham, MA, 31331028), supplemented with 10% fetal bovine serum (ThermoFisher, Waltham, MA, 10100139) and 5 μg/ml plasmocin™ (InvivoGen, San Diego, CA, ant-mpt). The culture medium of SIM-A9 cells was supplemented with 5% horse serum (Sigma-Aldrich, Saint Louis, MO, H1270). Embryonic cortical-hippocampal neurons from wild-type (WT) and SREBF-2 mice (B6;SJL-Tg(rPEPCKSREBF2)788Reh/J, RRID:IMSR_JAX:003311) were isolated on day 16–17 of pregnancy by trypsin digestion following a standard protocol [31 (link)]. Dissociated cells were grown in Neurobasal™ medium (ThermoFisher, 21103–049) supplemented with 2.5% (v/v) B27 supplement (ThermoFisher, 17504–001), 0.5 mM L-glutamine (Sigma-Aldrich, G7513) and 5 μg/ml plasmocinTM (InvivoGen, ant-mpt), and plated onto poly-D-lysine (Sigma-Aldrich, P6407)- and laminin (Sigma-Aldrich, L2020)-coated plates at a density of 2 × 105 cells/cm2. Half of the culture medium was changed every 3 or 4 days. Over 95% of neuronal purity was confirmed by immunochemistry using antibodies targeting neuronal and glial markers. Experiments were performed at 7 to 10 days in vitro (DIV). All procedures involving animals and their care were approved by the ethics committee of the University of Barcelona and were conducted in accordance with institutional guidelines in compliance with national and international laws and policies.
Cell cholesterol enrichment was achieved by incubation with a cholesterol:methyl-β-cyclodextrin complex (CHO:MCD; containing 50 μg/ml of cholesterol) (Sigma-Aldrich, C4951) for 1 h followed by 4-h recovery. To induce inflammasome activation, cells were treated with 10 μg/ml lipopolysaccharide (LPS) from Escherichia coli O111:B4 (Sigma-Aldrich, L4391), 10 μg/ml N-acetylmuramyl-L-alanyl-D-isoglutamine hydrate (also known as muramyl dipeptide, MDP; Sigma-Aldrich, A9519), 5 mM ATP (Sigma-Aldrich, A2383), 150 μg/ml monosodium urate crystals (MSU; Santa Cruz Biotech., sc-202711), and oligomeric Aβ at the indicated times. Preincubation with 4 mM glutathione ethyl ester (GSHee) or with the cell-permeable caspase 1 inhibitor I (10 μM; Bachem, 4095744) was performed 30 min before treatment when indicated.
de Dios C., Abadin X., Roca-Agujetas V., Jimenez-Martinez M., Morales A., Trullas R., Mari M, & Colell A. (2023). Inflammasome activation under high cholesterol load triggers a protective microglial phenotype while promoting neuronal pyroptosis. Translational Neurodegeneration, 12, 10.
Full text: Click here
Publication 2023
Acetylmuramyl-Alanyl-Isoglutamine Animals Antibodies Cell Lines Cells Cholesterol Cortex, Cerebral Culture Media Cyclodextrins Dietary Supplements Digestion Embryo Equus caballus Escherichia coli Ethics Committees Fetal Bovine Serum Glutamine Homo sapiens Inflammasomes interleukin-1beta-converting enzyme inhibitor isoglutamine Laminin Lipopolysaccharides Lysine Microglia Mus Neuroblastoma Neuroglia Neurons Permeability plasmocin Poly A Pregnancy S-ethyl glutathione Serum Sodium Urate Monohydrate Trypsin
3
NLRP3-Inflammasome Expression Plasmids
To express the components of the NLRP3-inflammasome (NLFP3-I), we utilized four plasmids expressing mouse NLRP3 (pcDNA3-N-Flag-NLRP3, Addgene plasmid # 75127), ASC (pcDNA3-N-Flag-ASC1, Addgene plasmid # 75134), CASP1 (pcDNA3-N-Flag-Caspase-1, Addgene plasmid # 75128) and pro-IL-1B (pCMV-pro-Il1b-C-Flag, Addgene plasmid # 75131). The use and construction of the NLRP3-I expression plasmids were previously described (25 (link)).
The plasmid vector used to express mitochondrial-targeted catalase (mCAT) and its respective control vector were p-mCAT (VectorBuilder ID VB170403-1078nzg) and p-EV (VectorBuilder ID VB210726-1273jte). The p-mCAT transgene cassette was transcribed from the 212 nucleotide elongation factor α1 “short” (EFS) promoter. The EFS promoter transcribes a polycistronic transcript encoding EGFP (enhanced green fluorescent protein), a self-cleaving 2A peptide site, followed by mCAT cDNA, and terminated by a simian virus 40 (SV40) late polyA sequence. p-EV is identical to the p-mCAT construct, except lacking the EGFP and mCAT sequences.
Guarnieri J.W., Angelin A., Murdock D.G., Schaefer P., Portluri P., Lie T., Huang J, & Wallace D.C. (2023). SARS-COV-2 viroporins activate the NLRP3-inflammasome by the mitochondrial permeability transition pore. Frontiers in Immunology, 14, 1064293.
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Publication 2023
Caspase 1 Catalase Cloning Vectors DNA, Complementary Elongation Factor enhanced green fluorescent protein Inflammasomes Interleukin-1 Mitochondria Nod-like receptor protein 3 inflammasome, mouse Nucleotides Peptides Plasmids Poly A Simian virus 40 Transgenes
4
Evaluating Immune Modulation in Giardia Infection
Mice were classified into two groups: (i) the MOCK control group and (ii) the MCC950 inhibitor group. In each group, there were five treatment methods (n = 7/treatment group): (i) the PBS treatment negative control group (PBS only; 100 µl/mouse PBS through intramuscular (i.m.) injection (anterior tibial muscle) [28 (link), 29 (link)]; (ii) pcDNA3.1(+) plasmid negative control group (100 µg/mouse DNA through i.m. injection); (iii) G. duodenalis cyst infection positive control group (1.5 × 106 cysts/mouse by gavage); (iv) pcDNA3.1(+)-alpha-2 plasmid treatment group (100 µg/mouse DNA through i.m. injection); and (v) pcDNA3.1(+)-alpha-7.3 plasmid treatment group (100 µg/mouse DNA through i.m. injection). After 12 h, the mice in the MCC950 inhibitor group received MCC950 (10 mg/kg BW) i.p. daily for 7 days, whereas the mice in the MOCK group were treated with an equal volume of PBS. Blood samples were collected from the eyeballs of the mice and allowed to stand overnight at 4 °C. Serum samples were separated by enzyme-linked immunosorbent assay (ELISA) to measure the IL-1β levels.
Zhao P., Li J., Li X., Dong J., Wang X., Zhang N., Li S., Sun M., Zhang X., Wang Z., Liang M., Li Y., Cao L, & Gong P. (2023). The NLRP3 inflammasome recognizes alpha-2 and alpha-7.3 giardins and decreases the pathogenicity of Giardia duodenalis in mice. Parasites & Vectors, 16, 85.
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Publication 2023
BLOOD Cyst Enzyme-Linked Immunosorbent Assay Eye Infection Infection Control Inflammasomes Interleukin-1 beta MCC-950 Mice, House Mus Plasmids Serum Tibial Muscle, Anterior Tube Feeding
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Giardia Infection and NLRP3 Inhibition
Mice were divided into four groups (n = 7 per group): (i) PBS treatment negative control group (PBS only; 100 µl/mouse PBS by gavage, followed 3 h later by 100 µl/mouse PBS i.p. route daily for 7 days); (ii) MCC950 inhibitor [27 (link)] treatment negative control group (100 µl/mouse PBS by gavage, followed 3 h later by 10 mg/kg body weight [BW] MCC950 [in PBS] by i.p. route daily for 7 days); (iii) G. duodenalis cyst infection group (1.5 × 106 cysts/mouse by gavage, followed 3 h later by 100 µl/mouse PBS by i.p. route daily for 7 days); and (iv) G. duodenalis cyst infection combined with group MCC950 inhibitor treatment group (1.5 × 106 cysts/mouse by gavage, followed 3 h later by 10 mg/kg BW MCC950 i.p. route daily for 7 days). The BW of each mouse was monitored daily, and all mice were euthanized on the 7th day. The harvested duodenum (3 cm long) was cut into small pieces in 1 ml of PBS, the cysts were disrupted in PBS at 4 °C overnight and the number of G. duodenalis trophozoites was counted under an optical microscope (Nikon Corp., Japan). Fresh duodenum (1 cm long) was isolated for hematoxylin and eosin (H&E) staining.
Zhao P., Li J., Li X., Dong J., Wang X., Zhang N., Li S., Sun M., Zhang X., Wang Z., Liang M., Li Y., Cao L, & Gong P. (2023). The NLRP3 inflammasome recognizes alpha-2 and alpha-7.3 giardins and decreases the pathogenicity of Giardia duodenalis in mice. Parasites & Vectors, 16, 85.
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Publication 2023
Body Weight Combined Modality Therapy Cyst Duodenum Eosin Giardiasis Hematoxylin Infection Inflammasomes Light Microscopy MCC-950 Mice, House Trophozoite Tube Feeding

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