The hepatocyte cell line RALA255-10G was cultured under nontransformed conditions, as described previously20 (link). Wild-type and Atg5−/− mouse embryonic fibroblasts provided by N. Mizushima21 (link) were cultured as described previously22 (link). TG content was determined by the Trig/GB Kit (Roche Diagnostics), cholesterol content by the Amplex Red Cholesterol Assay (Invitrogen), fatty acid β-oxidation by a modification of a previously used method23 (link), and TG decay in cells radiolabelled with [14C]oleate and TG synthesis by standard methods12 (link). shRNAs were cloned into pSUPER (Ambion) and then pCCL.sin.PPT.hPGK.GFPWpre24 (link). Protein isolation and western blotting were performed as described previously25 (link). Fluorescence microscopy for BODIPY 493/503 (Invitrogen) and immunofluorescence were performed as described previously26 (link). Atg7F/F mice4 (link) were crossed with Alb-Cre mice27 (link) to generate Atg7F/F-Alb-Cre mice. Some animals were fed a high-fat diet (60% kcal in fat; Research Diets, D12492). Electron microscopy and immunogold labelling were performed as described previously26 (link). LDs from mouse livers were isolated by sucrose density gradient centrifugation28 and autophagic vacuoles and lysosomes by centrifugation in metrizamide discontinuous density gradients29 (link).
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Anatomy
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Cell Component
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Lysosomes
Lysosomes
Lysosomes are specialized organelles found in eukaryotic cells that function as the cell's digestive system.
These membrane-bound vesicles contain a variety of hydrolytic enzymes capable of breaking down a wide range of biological molecules, including proteins, nucleic acids, carbohydrates, and lipids.
Lysosomes play a crucial role in cellular homeostasis, recycling, and the degradation of damaged or unwanted cellular components.
Dysfunctions in lysosomal structure or activity have been implicated in a number of inherited metabolic disorders, known as lysosomal storage diseases, as well as in more common age-related diseases like Alzheimer's and Parkinson's.
Understanding the mechanisms governing lysosome function is an active area of research with important implications for human health and disease.
A typo: Lysosomes are specialzied organelles found in eukaryotic cells that function as the cell's digestive system.
These membrane-bound vesicles contain a variety of hydrolytic enzymes capable of breaking down a wide range of biological molecules, including proteins, nucleic acids, carbohydrates, and lipids.
Lysosomes play a crucial role in cellular homeostasis, recycling, and the degradation of damaged or unwanted cellular components.
Dysfunctions in lysosomal structure or activity have been implicated in a number of inherited metabolic disorders, known as lysosomal storage diseases, as well as in more common age-related diseases like Alzheimer's and Parkinson's.
Understanding the mechanisms governing lysosome function is an active area of research with important implications for human health and disease.
A typo: Lysosomes are specialzied organelles found in eukaryotic cells that function as the cell's digestive system.
Most cited protocols related to «Lysosomes»
4,4-difluoro-1,3,5,7,8-pentamethyl-4-bora-3a,4a-diaza-s-indacene
Anabolism
Animals
Autophagosome
Biological Assay
Cell Lines
Cells
Centrifugation
Cholesterol
Diagnosis
Diet
Diet, High-Fat
Electron Microscopy
Embryo
Fatty Acids
Fibroblasts
formaldehyde-serum albumin
Hepatocyte
Immunofluorescence
isolation
Liver
Lysosomes
Metrizamide
Mice, Laboratory
Microscopy, Fluorescence
Oleate
Proteins
Short Hairpin RNA
Sucrose
Animals
Biological Assay
Cell Culture Techniques
Cells
Cultured Cells
Hepatocyte
Homozygote
isolation
Liver
Lysosomes
Proteolysis
Tissues
To complement incomplete annotations in the background database, a homology-ontology annotation retrieved by BLAST should be accompanied by an accurate subcellular localization prediction for each homologous sequence. CELLO has been shown to be helpful for the prediction of subcellular localizations of the proteins found in a proteomic data. [28] (link) Using multiple, integrated machine-learned classifiers, CELLO predicts which of four subcellular localizations in archaea and in Gram-positive bacteria, five subcellular localizations in Gram-negative bacteria, and twelve subcellular localizations in eukaryotes that the targeted protein might be found in, with the four archaeal and Gram-positive bacterial localizations being the extracellular space, the cell wall, the cytoplasmic membrane, and the cytoplasm; the five Gram-positive bacterial localizations being the extracellular space, the outer membrane, the periplasmic and cytoplasmic (inner) membranes, and the cytoplasm; and the 12 eukaryotic localizations being chloroplasts, the cytoplasm, the cytoskeleton, the endoplasmic reticulum, the extracellular/secretory space, the Golgi, lysosomes, mitochondria, the nucleus, peroxisomes, the plasma membrane, and vacuoles. Due to subcellular data increased exponentially over the years, CELLO has been trained on latest models and denoted as update version wrapping in CELLO2GO. And the resultant datasets used for prediction and evaluation is from PSORTb3.0 [23] (link).
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Archaea
Cell Nucleus
Cell Wall
Chloroplasts
Cytoplasm
Cytoskeleton
Endoplasmic Reticulum
Eukaryota
Eukaryotic Cells
Extracellular Space
Golgi Apparatus
Gram-Positive Bacteria
Gram Negative Bacteria
Homologous Sequences
Lysosomes
Mitochondria
Periplasm
Peroxisome
Plasma Membrane
Proteins
secretion
Tissue, Membrane
Vacuole
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.
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
Antibodies
Biological Assay
Cell Death
Cell Degranulation
Cell Lines
Cells
Chemokine
Cytokine
Cytotoxin
Differential Diagnosis
Homo sapiens
Hypersensitivity
Interferon Type II
Kinetics
lysosomal proteins
Lysosomes
Natural Killer Cells
PBMC Peripheral Blood Mononuclear Cells
Plasma Membrane
Protein Biosynthesis
Protoplasm
secretion
Secretions, Bodily
Secretory Granule
Transcription, Genetic
Tumor Necrosis Factor-alpha
Most recents protocols related to «Lysosomes»
Example 4
To determine where 2F2-grafted “humanized” antibodies and antibody variants are delivered upon internalization into the cell, colocalization studies of the anti-CD79b antibodies internalized into B-cell lines may be assessed in Ramos cell lines. LAMP-1 is a marker for late endosomes and lysosomes (Kleijmeer et al., Journal of Cell Biology, 139(3): 639-649 (1997); Hunziker et al., Bioessays, 18:379-389 (1996); Mellman et al., Annu. Rev. Dev. Biology, 12:575-625 (1996)), including MHC class II compartments (MIICs), which is a late endosome/lysosome-like compartment. HLA-DM is a marker for MIICs.
Ramos cells are incubated for 3 hours at 37° C. with 1 μg/ml 2F2-grafted “humanized” antibodies and antibody variants, FcR block (Miltenyi) and 25 μg/ml Alexa647-Transferrin (Molecular Probes) in complete carbonate-free medium (Gibco) with the presence of 10 μg/ml leupeptin (Roche) and 5 μM pepstatin (Roche) to inhibit lysosomal degradation. Cells are then washed twice, fixed with 3% paraformaldehyde (Electron Microscopy Sciences) for 20 minutes at room temperature, quenched with 50 mM NH4Cl (Sigma), permeabilized with 0.4% Saponin/2% FBS/1% BSA for 20 minutes and then incubated with 1 μg/ml Cy3 anti-mouse (Jackson Immunoresearch) for 20 minutes. The reaction is then blocked for 20 minutes with mouse IgG (Molecular Probes), followed by a 30 minute incubation with Image-iT FX Signal Enhancer (Molecular Probes). Cells are finally incubated with Zenon Alexa488-labeled mouse anti-LAMP1 (BD Pharmingen), a marker for both lysosomes and MIIC (a lysosome-like compartment that is part of the MHC class II pathway), for 20 minutes, and post-fixed with 3% PFA. Cells are resuspended in 20 μl saponin buffer and allowed to adhere to poly-lysine (Sigma) coated slides prior to mounting a coverglass with DAPI-containing VectaShield (Vector Laboratories). For immunofluorescence of the MIIC or lysosomes, cells are fixed, permeabilized and enhanced as above, then co-stained with Zenon labeled Alexa555-HLA-DM (BD Pharmingen) and Alexa488-Lamp1 in the presence of excess mouse IgG as per the manufacturer's instructions (Molecular Probes).
Accordingly, colocalization of 2F2-grafted “humanized” antibodies or antibody variants with MIIC or lysosomes of B-cell lines as assessed by immunofluorescence may indicate the molecules as excellent agents for therapy of tumors in mammals, including B-cell associated cancers, such as lymphomas (i.e. Non-Hodgkin's Lymphoma), leukemias (i.e. chronic lymphocytic leukemia), and other cancers of hematopoietic cells.
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Alexa Fluor 647
Anti-Antibodies
Antibodies, Monoclonal, Humanized
B-Lymphocytes
Buffers
Carbonates
CD79B protein, human
Cell Lines
Cells
Chronic Lymphocytic Leukemia
Cloning Vectors
DAPI
Electron Microscopy
Endosomes
Genes, MHC Class II
Hematopoietic Neoplasms
Immunofluorescence
Immunoglobulins
Leukemia
leupeptin
Lymphoma
Lymphoma, Non-Hodgkin
Lysine
lysosomal-associated membrane protein 1, human
Lysosomes
Malignant Neoplasms
Mammals
Molecular Probes
Mus
Neoplasms
paraform
pepstatin
Poly A
Saponin
Therapeutics
Transferrin
Example 10
The ETSD design successfully translocated N to the endosomal subcellular compartment. After infection of HeLa cells with N-ETSD, N co-localized with the endosomal marker 45 transferrin receptor (CD71), as shown in
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Cytoplasm
Endosomes
HeLa Cells
Infection
lysosomal-associated membrane protein 1, human
Lysosomes
TFRC protein, human
Example 6
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Chitosan
Fibrosis
Lysosomes
Phosphates
Saline Solution
Solvents
Sutures
Tissue, Membrane
Example 11
Based on the evidence that S-Fusion+N-ETSD resulted in enhanced expression of physiologically-relevant RBD and that N-ETSD successfully translocated to the endosomal/lysosomal compartment, the bivalent hAd5 S-Fusion+N-ETSD vaccine was chosen for inoculation of 7-week old female CD-1 mice. The unique properties of this construct would result in the generation of both CD8+ and CD4+ T-cell responses and neutralizing antibodies. As described in Methods, mice received an initial injection on Day 0 and a second injection on Day 21. Sera were collected on Day 0 and at the end of the study on Day 28 for antibody and neutralization analyses. Splenocytes were also collected on Day 28 for intracellular cytokine staining (ICS) and ELISpot analyses. All age- and gender-matched animals assigned to the study appeared normal with no site reactions and no loss of body weight throughout the dosing were seen, consistent with previous observations with the hAd5 [E1-, E2b-, E3-] platform
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Animals
Antibodies, Neutralizing
Binding Sites
CD4 Positive T Lymphocytes
Cytokine
Endosomes
Enzyme-Linked Immunospot Assay
Females
Human Body
Immunogenicity, Vaccine
Immunoglobulins
Lysosomes
Mus
Protoplasm
Serum
Vaccination
Vaccines
Vision
Knockout (KO) of GlcNAc-1-phosphate transferase subunits alpha and beta (Gnptab) gene was carried out in selected CHO clones that stably express the lysosomal enzymes of interest. Cells were seeded at a density of 0.5 × 106 cells/mL in T25 flasks 24 h prior to transfection, and ∼2 × 106 cells and 1 μg each of plasmid DNA of Cas9-GFP and gRNA were used for electroporation. 48 h after electroporation, cells with GFP expression were enriched by FACS. After culturing for 1 week, cells were single cell sorted by FACS into 96-wells. KO clones with desired mutations were identified using a rapid and efficient screening method, Indel Detection by Amplicon Analysis (IDAA), as previously described (Yang et al., 2015a (link)). Final clones were verified by Sanger sequencing. On average 2–5 clones with frameshift mutations were selected from each targeting event. The full list of CRISPR gRNA design and PCR primers used is listed elsewhere (Tian et al., 2019 (link)).
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Cells
Clone Cells
Clustered Regularly Interspaced Short Palindromic Repeats
Electroporation
Enzymes
Frameshift Mutation
Gene Knockout Techniques
Glycoprotein Hormones, alpha Subunit
INDEL Mutation
Lysosomes
Mutation
N-acetylglucosamine-1-phosphate
Oligonucleotide Primers
Plasmids
Transfection
Transferase
Top products related to «Lysosomes»
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LysoTracker Red DND-99 is a fluorescent dye that selectively stains acidic organelles, such as lysosomes, in live cells. It can be used to visualize and monitor the distribution and activity of lysosomes within the cellular environment.
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LysoTracker Red is a fluorescent dye that selectively stains acidic organelles, such as lysosomes, in live cells. It is a useful tool for visualizing and tracking the dynamics of these organelles within the cellular environment.
Sourced in United States, United Kingdom, Japan, Germany
LysoTracker is a fluorescent dye that selectively labels acidic organelles, such as lysosomes, in live cells. It is a useful tool for the visualization and analysis of lysosomal function in various cell types and biological applications.
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LysoTracker Green DND-26 is a fluorescent probe that selectively stains acidic organelles, such as lysosomes, in live cells. It is a membrane-permeant dye that accumulates in acidic compartments due to protonation. The dye exhibits green fluorescence upon uptake, allowing visualization and tracking of lysosomal dynamics.
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Hoechst 33342 is a fluorescent dye that binds to DNA. It is commonly used in various applications, such as cell staining and flow cytometry, to identify and analyze cell populations.
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Bafilomycin A1 is a macrolide compound that acts as a potent and specific inhibitor of vacuolar-type H+-ATPases (V-ATPases). V-ATPases are involved in the acidification of various intracellular compartments, making Bafilomycin A1 a useful tool for studying cellular processes that rely on pH regulation.
Sourced in United States, United Kingdom
LysoTracker Deep Red is a fluorescent dye that selectively accumulates in the acidic compartments of live cells, such as lysosomes. It is useful for labeling and tracking the dynamics of these organelles in various cellular processes.
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MG132 is a proteasome inhibitor, a type of laboratory reagent used in research applications. It functions by blocking the activity of the proteasome, a complex of enzymes responsible for the degradation of proteins within cells. MG132 is commonly used in cell biology and biochemistry studies to investigate the role of the proteasome in various cellular processes.
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Chloroquine is a laboratory chemical primarily used as a research tool in biochemical and cell biology applications. It is a white, crystalline solid that is soluble in water. Chloroquine is commonly used in experiments to study cellular processes, such as autophagy and endocytosis, by inhibiting the function of lysosomes. Its core function is to serve as a research reagent for scientific investigations, without making any claims about its intended use.
Sourced in United States, Germany, United Kingdom, Japan
LysoTracker Green is a fluorescent probe that selectively labels acidic organelles, such as lysosomes, in live cells. It is a useful tool for monitoring lysosomal function and dynamics.
More about "Lysosomes"
Lysosomes are specialized, membrane-bound organelles found in eukaryotic cells that function as the cell's digestive system.
These organelles contain a variety of hydrolytic enzymes capable of breaking down a wide range of biological molecules, including proteins, nucleic acids, carbohydrates, and lipids.
Lysosomes play a crucial role in cellular homeostasis, recycling, and the degradation of damaged or unwanted cellular components.
Dysfunctions in lysosomal structure or activity have been implicated in a number of inherited metabolic disorders, known as lysosomal storage diseases, as well as in more common age-related diseases like Alzheimer's and Parkinson's.
Understanding the mechanisms governing lysosome function is an active area of research with important implications for human health and disease.
Lysosome research often utilizes fluorescent dyes like LysoTracker Red DND-99, LysoTracker Red, LysoTracker, LysoTracker Green DND-26, and LysoTracker Deep Red to visualize and study these organelles.
Other compounds like Hoechst 33342, Bafilomycin A1, and Chloroquine are also used to investigate lysosomal structure and function.
The proteasome inhibitor MG132 can also provide insights into the role of lysosomes in protein degradation.
Optimizing research protocols and accurately locating the best published methods is crucial for advancing our understanding of these important cellular structures.
PubCompare.ai's AI-powered tools can help researchers unlock reproducibility and accuracy in lysosome studies by effortlessly comparing published protocols and identifying the most suitable approaches.
These organelles contain a variety of hydrolytic enzymes capable of breaking down a wide range of biological molecules, including proteins, nucleic acids, carbohydrates, and lipids.
Lysosomes play a crucial role in cellular homeostasis, recycling, and the degradation of damaged or unwanted cellular components.
Dysfunctions in lysosomal structure or activity have been implicated in a number of inherited metabolic disorders, known as lysosomal storage diseases, as well as in more common age-related diseases like Alzheimer's and Parkinson's.
Understanding the mechanisms governing lysosome function is an active area of research with important implications for human health and disease.
Lysosome research often utilizes fluorescent dyes like LysoTracker Red DND-99, LysoTracker Red, LysoTracker, LysoTracker Green DND-26, and LysoTracker Deep Red to visualize and study these organelles.
Other compounds like Hoechst 33342, Bafilomycin A1, and Chloroquine are also used to investigate lysosomal structure and function.
The proteasome inhibitor MG132 can also provide insights into the role of lysosomes in protein degradation.
Optimizing research protocols and accurately locating the best published methods is crucial for advancing our understanding of these important cellular structures.
PubCompare.ai's AI-powered tools can help researchers unlock reproducibility and accuracy in lysosome studies by effortlessly comparing published protocols and identifying the most suitable approaches.