PMN transepithelial migration and subsequent inflammation occur first in the small airways of CF patients, i.e., the bronchiolar region, which is lined with a microvilli-covered epithelial monolayer dominated by Club cells [3 (link)–5 (link)]. Therefore, to mimic PMN transmigration into the small airway lumen, we selected the H441 human Club cell line [20 (link)] to grow epithelial monolayers at air-liquid interface (ALI). To enable PMN loading in the lamina propria and transepithelial migration (Fig. 1A ), we used Alvetex (Reinnervate) 200 μm-thick inert 3D scaffolds with >90% porosity (pore sizes of 36–40 μm, with interconnects of 12–14 μm). In brief, inserts were activated with 70% ethanol, coated overnight at 37ºC with rat-tail collagen I (3 mg/mL, Sigma) and seeded with H441 cells at 2.5×105 cells per 12-well insert. Cells were first grown in submerged cultures with DMEM/F12 supplemented with 10% heat-inactivated serum, penicillin, and streptomycin. After 2 days, cells were supplemented basally with serum-free DMEM/F12 with 10% Ultroser G (Pall Life Sciences) to establish ALI. Cultures were grown for 2 weeks at ALI and supplemented basally with fresh medium every 48 hours. For TM experiments, the ALI cultures were placed with the apical compartment exposed to RPMI, leukotriene B4 (LTB4, 100 nM), CXCL8 (100 ng/mL), formyl-methionine-leucine-phenylalanine (fMLF, 100 ng/mL), lipopolysaccharide (LPS, 500 ng/mL), or airway supernatant (ASN) from CF, HC, COPD, and LD subjects. TM experiments with 0.5–1 ×106 PMNs loaded onto the 200 μm-thick basal compartment of the Alvetex scaffold (situated upside), and allowed to migrate at 37°C at 5% CO2 through the collagen and epithelial layers into the apical compartment (situated downside, and bathed with either control medium with chemoattractant, or ASN). In some experiments, drugs were added to apical ASN and/or basal PMN suspensions. In other experiments, LPS-RS (competitive inhibitor of LPS binding to TLR4) was added to apical LPS or CF ASN. LPS and LPS-RS were purchased as ultrapure reagents from InvivoGen.
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Anatomy
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Cell Component
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Microvilli
Microvilli
Microvilli are small, finger-like projections that extend from the apical surface of certain epithelial cells, such as those found in the intestinal lining.
These structures play a crucial role in increasing the surface area of the cell, allowing for more efficient absorption and transport of nutrients, ions, and other molecules.
Microvilli are composed of a core of actin filaments and are covered by the cell membrane, which is continuous with the cell's surface.
Studying the structure, function, and regulation of microvilli is an important area of research in fields like cell biology, physiology, and digestive system disorders.
Researchers can leverage PubCompare.ai's AI-driven protocol comparisons to optimize their microvilli research workflows, locate the best methods from literature, preprints, and patents, and improve reproducibility and accuracy.
These structures play a crucial role in increasing the surface area of the cell, allowing for more efficient absorption and transport of nutrients, ions, and other molecules.
Microvilli are composed of a core of actin filaments and are covered by the cell membrane, which is continuous with the cell's surface.
Studying the structure, function, and regulation of microvilli is an important area of research in fields like cell biology, physiology, and digestive system disorders.
Researchers can leverage PubCompare.ai's AI-driven protocol comparisons to optimize their microvilli research workflows, locate the best methods from literature, preprints, and patents, and improve reproducibility and accuracy.
Most cited protocols related to «Microvilli»
Bronchioles
Cell Lines
Cells
Chemotactic Factors
Chronic Obstructive Airway Disease
Collagen
Collagen Type I
CXCL8 protein, human
Ethanol
Homo sapiens
Inflammation
Lamina Propria
Leucine
Leukotriene B4
Microvilli
N-Formylmethionine
Patients
Penicillins
Pharmaceutical Preparations
Phenylalanine
Serum
Streptomycin
Tail
Transepithelial Migration
Ultroser G
To screen for potentpharmaceutical compounds from Sanhe Decoction, an in silico ADME-systems evaluation model, which integrated drug-likeness (DL), oral bioavailability (OB), aqueous solubility (logS, the logarithm of aqueous solubility), lipophilicity (logP, logarithm of octanol-water partition coefficient) and Caco-2 permeability was proposed.
Lipophilicity: The lipophilicity was expressed as the partition coefficient P (log P), which is calculated by ALOGPS 2.1 software128 (link). The value of log P less than 5 was selected for further analysis.
Aqueous solubility: Log S, a measure of aqueous solubility, which has been considered as an important factor in drug absorption and distribution. The value of Log S is also calculated by ALOGPS 2.1 software128 (link) and the threshold value is range from −5 to −1.
Drug-likeness: To filter out the drug-like molecules, we have developed a database-dependent model to discriminate between drug-like and nondrug-like chemicals using the Tanimoto coefficient129 . This model is constructed based on the molecular descriptors and Tanimoto coefficient (as displayed inEquation2 ).
where A is the molecular properties of herbal ingredients, and B represents the average molecular properties of molecules in DrugBank database (http://www.drugbank.ca/ ) based on Dragon soft descriptors130 . In this work, the molecules with satisfying drug-likeness index (DL ≥ 0.18) (average value for Drugbank) were selected as candidate compounds.
Oral bioavailability: OB, represents the rate and extent to which the active ingredient or active moiety is absorbed from a drug product and becomes available at the site of action131 (link). Here the OB screening was performed by an in-house system OBioavail1.131 (link) and the compounds with OB ≥ 30% were kept in the database.
Caco-2 permeability: For an orally administered drug, the majority of drug absorption occurs in the small intestine where the presence of villi and microvilli greatly increases the surface available for absorption132 (link). Here, we employed a robust in silico Caco-2 permeability prediction model PreCaco2133 to predict the drug absorption. We set the threshold of Caco-2 permeability to −0.4, because the compounds with Caco-2 value less than −0.4 are considered to be not permeable.
Lipophilicity: The lipophilicity was expressed as the partition coefficient P (log P), which is calculated by ALOGPS 2.1 software128 (link). The value of log P less than 5 was selected for further analysis.
Aqueous solubility: Log S, a measure of aqueous solubility, which has been considered as an important factor in drug absorption and distribution. The value of Log S is also calculated by ALOGPS 2.1 software128 (link) and the threshold value is range from −5 to −1.
Drug-likeness: To filter out the drug-like molecules, we have developed a database-dependent model to discriminate between drug-like and nondrug-like chemicals using the Tanimoto coefficient129 . This model is constructed based on the molecular descriptors and Tanimoto coefficient (as displayed in
where A is the molecular properties of herbal ingredients, and B represents the average molecular properties of molecules in DrugBank database (
Oral bioavailability: OB, represents the rate and extent to which the active ingredient or active moiety is absorbed from a drug product and becomes available at the site of action131 (link). Here the OB screening was performed by an in-house system OBioavail1.131 (link) and the compounds with OB ≥ 30% were kept in the database.
Caco-2 permeability: For an orally administered drug, the majority of drug absorption occurs in the small intestine where the presence of villi and microvilli greatly increases the surface available for absorption132 (link). Here, we employed a robust in silico Caco-2 permeability prediction model PreCaco2133 to predict the drug absorption. We set the threshold of Caco-2 permeability to −0.4, because the compounds with Caco-2 value less than −0.4 are considered to be not permeable.
compound 30
Intestines, Small
Microvilli
Octanols
Permeability
Pharmaceutical Preparations
2 d postinjection, oocyte swelling assays were performed at room temperature (20–21°C) by transfer from 200 mosM (100% ND96) to 70 mosM (30% ND96) solution as previously described (Németh-Cahalan and Hall, 2000 (link)). Water permeability, Pf, was calculated from optical measurements of the increase in cross-sectional area of the oocyte with time in response to diluted ND96 using the formula: where V is the volume as a function of time, V0 is the initial volume, S is the geometric surface area, Δosm is the osmotic gradient, and Vw is the molar volume of water. Previously (Chandy et al., 1997 (link); Németh Cahalan and Hall, 2000 (link)), we reported water permeabilities using the actual surface area of the oolemma, which is nine times the geometric surface area due to folds and microvilli as determined by morphological measurement and by oocyte membrane capacitance (Dick and Dick, 1970 (link); Zampighi et al., 1995 (link)). To facilitate comparison with results in the bulk of the literature, the permeabilities reported here are calculated using the geometric surface area. The swelling assay is performed either under control conditions of pH 7.5 and 1.8 mM Ca2+, or experimental conditions of altered pH or Ca2+. Unless otherwise indicated in parentheses, each data point is the average of measurements from nine oocytes from three different batches.
Biological Assay
Dietary Fiber
Microvilli
Molar
Oocytes
Osmosis
Permeability
Tissue, Membrane
At the end of 6 weeks trial, 6 fish per treatment were selected randomly and euthanized with an overdose of AQUI-S® to excise liver, intraperitoneal fat and intestine for histological analysis. Fragments of all tissue samples were fixed in 10% neutral buffered formalin, dehydrated with a series of ethanol concentrations before infiltrating in xylene and embedding in paraffin wax, and finally sectioned at approximately 5 µm using a rotary microtome machine for staining with hematoxylin and eosin (H&E) following the standard histological procedures. Histological slides were digitally photographed under a light imaging microscope (BX40F4, Olympus, Tokyo, Japan).
Intestinal section were further subjected to two different stains including Periodic Acid-Schiff (PAS) and Alcian Blue (AB) pH 2.5 staining to identify neutral mucins and acidic mucins, respectively. Both type of mucins were counted from ten intact randomly selected villi, as described earlier by Elia, et al.1 (link). Ten intact villi were randomly selected to measure the intestine histometric in terms of villi height and width, enterocyte width, muscular wall, submucosa thickness and microvilli height and diameters of adipocyte were measured using ImageJ software.
Intestinal section were further subjected to two different stains including Periodic Acid-Schiff (PAS) and Alcian Blue (AB) pH 2.5 staining to identify neutral mucins and acidic mucins, respectively. Both type of mucins were counted from ten intact randomly selected villi, as described earlier by Elia, et al.1 (link). Ten intact villi were randomly selected to measure the intestine histometric in terms of villi height and width, enterocyte width, muscular wall, submucosa thickness and microvilli height and diameters of adipocyte were measured using ImageJ software.
Acids
Adipocytes
Alcian Blue
Drug Overdose
Enterocytes
Eosin
Ethanol
Fishes
Formalin
Hematoxylin
Histological Techniques
Intestines
Light Microscopy
Liver
Microtomy
Microvilli
Mucins
Muscle Tissue
Periodic Acid
Stains
Tissues
Xylene
The segments of the jejunum and ileum fixed in 4 % formaldehyde were used to determine morphology using hematoxylin-eosin staining. After dehydration, embedding, sectioning, and staining, images were acquired at various magnifications with computer-assisted microscopy (Micrometrics TM; Nikon ECLIPSE E200, Tokyo, Japan). Villous height, crypt depth, goblet cell and lymphocyte counts were measured by Image-Pro Plus software, Version 6.0 on images at 200- or 400-fold magnification in five randomly selected fields, respectively [4 (link)].
Segments of the jejunum and ileum at higher magnification were also designated for analysis by scanning electron microscopy as described by German [16 (link)] and Liu et al. [17 (link)]. Briefly, tissue segments were fixed with 2.5 % glutaraldehyde for 2 h at 4 °C, and rinsed 3 × 10 min in PBS at 4 °C. The tissues were then postfixed in 1 % osmium tetroxide for 12 h at 4 °C, and rinsed 3 × 10 min in PBS at 4 °C. After dehydration in a graded ethanol series, the tissues stored in tert butyl alcohol for 2 h at room temperature. After samples were mounted onto stubs by means of quick-drying silver paint, the tissues were coated with gold-palladium and examined by a JEOL JSM-6360LV scanning electron microscope at 25 KV. The apparent characteristics of microvillus were observed and described.
Segments of the jejunum and ileum at higher magnification were also designated for analysis by scanning electron microscopy as described by German [16 (link)] and Liu et al. [17 (link)]. Briefly, tissue segments were fixed with 2.5 % glutaraldehyde for 2 h at 4 °C, and rinsed 3 × 10 min in PBS at 4 °C. The tissues were then postfixed in 1 % osmium tetroxide for 12 h at 4 °C, and rinsed 3 × 10 min in PBS at 4 °C. After dehydration in a graded ethanol series, the tissues stored in tert butyl alcohol for 2 h at room temperature. After samples were mounted onto stubs by means of quick-drying silver paint, the tissues were coated with gold-palladium and examined by a JEOL JSM-6360LV scanning electron microscope at 25 KV. The apparent characteristics of microvillus were observed and described.
CFC1 protein, human
Dehydration
Eosin
Ethanol
Formaldehyde
Glutaral
Goblet Cells
Gold
Ileum
Jejunum
Lymphocyte Count
Microscopy
Microvilli
Osmium Tetroxide
Palladium
Scanning Electron Microscopy
Silver
tert-Butyl Alcohol
Tissues
Most recents protocols related to «Microvilli»
Samples of mid-ileal tissue were collected at necropsy and rinsed with Dulbecco's phosphate buffered saline (D-PBS) at pH 7.4 (Sigma-Aldrich, St. Louis, MO). The luminal side was placed on top of a liver section with a thin layer of Tissue-Tek optimum cutting temperature (OCT) compound (Sakura Finetek, Torrance, CA) interface to protect microvilli structure from mechanical and environmental disruption. This also aided in determining tissue orientation upon cryosectioning. Tissue samples were then wrapped in aluminum foil and snap-frozen by placement in a tin cup of cold isopentane (Sigma-Aldrich) housed within a small cooler of dry ice and 95% ethanol slurry for a minimum of 5 min, then wrapped again in aluminum foil and transferred on dry ice to be stored at −80°C.
When cryosectioning was ready to be performed, tissue sections were removed from storage at −80°C and placed in a cryostat to acclimate to −20°C for at least 30 min. The entire tissue sample was embedded within Tissue-Tek optimal cutting temperature (O.C.T.; Sakura Finetek, Torrance, CA) and cut in 6 μm sections, then adhered to MAS slides (Matsunami Glass, Bellingham, WA). The tissue sections were dried overnight at room temperature and fixed in a 50% acetone/50% methanol solution for 5 min. Slides were stored at −80°C until ready for further processing.
When cryosectioning was ready to be performed, tissue sections were removed from storage at −80°C and placed in a cryostat to acclimate to −20°C for at least 30 min. The entire tissue sample was embedded within Tissue-Tek optimal cutting temperature (O.C.T.; Sakura Finetek, Torrance, CA) and cut in 6 μm sections, then adhered to MAS slides (Matsunami Glass, Bellingham, WA). The tissue sections were dried overnight at room temperature and fixed in a 50% acetone/50% methanol solution for 5 min. Slides were stored at −80°C until ready for further processing.
Acetone
Aluminum
Autopsy
Cold Temperature
Dry Ice
Ethanol
Freezing
Ileum
isopentane
Liver
Methanol
Microvilli
Phenobarbital
Phosphates
Saline Solution
Tissues
The plasma membrane mask from (section‐1/step‐1) usually contains small gaps at the cell surface, which may interfere with the downstream analysis. To close these gaps, we merged the output from (section‐1/step‐1) and the surface layer of the dome from (section‐1/step‐2). We subtracted the entire dome object except for its surface layer, from the plasma membrane mask, to ensure that the downstream analysis is confined to the cell surface and the microvilli, and that any other components within the cytoplasm are excluded from the analysis.
Cells
Cytoplasm
Microvilli
Plasma Membrane
The epithelial cell line 16HBE14o-, derived from human bronchial epithelial cells (Prof. D.C. Gruenert, University of California, San Francisco, CA, USA), was used as a surrogate for the respiratory epithelium. The cells show properties of differentiated airway epithelial cells including formation of tight junctions, apical microvilli and cilia and form polarized monolayers when grown on Transwell filters [44 (link)]. The 16HBE14o- cells were cultured at 37 °C in a humidified atmosphere containing 5% CO2 in Minimum Essential Medium (MEM; Gibco, Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (FBS; HyClone, GE Healthcare, Buckinghamshire, UK), 100 U/mL penicillin and 100 µg/mL streptomycin (Gibco, Thermo Fisher Scientific). Cells were cultured in flasks coated with LHC basal medium (Gibco, Thermo Fisher Scientific) supplemented with 100 µg/mL BSA, 30 µg/mL collagen and 10 µg/mL fibronectin (BD Biosciences, San Jose, CA, USA).
The cervical epithelial cell line H1HeLa (American Type Culture Collection, ATCC, Manassas, VA, USA) was grown at 37 °C in a humidified atmosphere containing 5% CO2 in MEM supplemented with 10% FBS (Gibco, Thermo Fisher Scientific) and antibiotics (100 U/mL penicillin, 100 µg/mL streptomycin and 1.2 µg/mL gentamycin (Gibco, Thermo Fisher Scientific).
The cervical epithelial cell line H1HeLa (American Type Culture Collection, ATCC, Manassas, VA, USA) was grown at 37 °C in a humidified atmosphere containing 5% CO2 in MEM supplemented with 10% FBS (Gibco, Thermo Fisher Scientific) and antibiotics (100 U/mL penicillin, 100 µg/mL streptomycin and 1.2 µg/mL gentamycin (Gibco, Thermo Fisher Scientific).
Antibiotics
Atmosphere
Bronchi
Cell Lines
Cells
Cilia
Collagen
Epithelial Cells
Fibronectins
Gentamicin
Homo sapiens
Microvilli
Neck
Penicillins
Respiratory Epithelium
Streptomycin
Tight Junctions
Human renal proximal tubular epithelial cells (PTCs) were isolated as described previously [49 (link)]. In brief, PTCs were separated after tumor nephrectomies from renal tissue not involved in cell carcinoma. Kidney tissue was minced by crossed blades, then digested with collagenase/dispase. The tissue was then filtered through a mesh (106 µm), redigested with collagenase IV, DNase, and MgCl2, and centrifuged using a Percoll density gradient. Finally, highly purified PTCs were separated immunomagnetically using a mAb against aminopeptidase M (CD13) and the Mini-MACS system (Miltenyi, Germany). Primary isolates were strongly positive for aminopeptidase M and ultrastructural analysis revealed well-preserved brush border microvilli, a well-developed endocytosis apparatus, and numerous mitochondria [49 (link),50 (link)]. PTCs were cultured in a standard culture medium (Medium 199 (M4530, Sigma, Taufkirchen, Germany) with a physiological glucose concentration (100 mg/dL) and with 10% fetal bovine serum (FBS; Biochrom, Berlin, Germany)). The medium was renewed every three to four days. Trypsinization was used to passage confluent cells, and passages 2 and 5 were used in this study. Cultured PTCs were characterized, as described previously [49 (link),50 (link)]. In addition, the SGLT-2 mRNA and protein expressions of cultured PTCs were shown by PCR analysis and immunofluorescence staining [12 (link)]. Glucose uptake in PTCs was shown by 2-Deoxy-2-[(7-nitro-2,1,3-benzoxadiazol-7-yl)amino]-D-glucose (NBDG-2), a fluorescent glucose analogon used in fluorescence measurements [12 (link)].
6-deoxy-N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)aminoglucose
Alanine Aminopeptidase
Aminopeptidase, Glutamyl
Brush Border
Carcinoma
Cells
Collagenase
Deoxyribonucleases
dispase
Endocytosis
Epithelial Cells
Factor IX Complex
Fluorescence
Fluorescent Antibody Technique
Glucose
Homo sapiens
Kidney
Magnesium Chloride
Microvilli
Mitochondria
Neoplasms
Nephrectomy
oxytocin, 1-desamino-(O-Et-Tyr)(2)-
Percoll
physiology
Proteins
RNA, Messenger
SLC5A2 protein, human
Tissues
Cellular tests were performed on the Caco-2 (ATCC® HTB-37™) human colorectal cancer cell line, which is a continuous line of heterogeneous human epithelial colorectal adenocarcinoma cells that, upon reaching confluence, express the characteristics of enterocytic differentiation: tight junctions, microvilli and a number of enzymes and transporters that are characteristic of enterocytes [18 (link)]. Caco-2 cells were grown in EMEM medium supplemented with 10% heat-inactivated fetal bovine serum (FBS), 2 mM L-glutamine, 0.1 mM non-essential amino acids, 100 U/mL penicillin, 100 µg/mL streptomycin. The Caco-2 cell line was maintained at 37 °C in a humidified 5% CO2 incubator. The ATCC cell line was validated by short tandem repeat profiles that are generated by simultaneous amplification of multiple short tandem repeat loci and amelogenin (for gender identification). All the reagents for cell cultures were supplied by Euroclone (Euroclone Spa, Milan, Italy). Caco-2 cells were seeded at a density of 1 × 104 cells/well in 96-well microtiter plates, at 1 × 106 cells/dish in 100 mm dish or at 90,000 cell/cm2 onto collagen coated Transwell® polyester membrane inserts, based on the suitable conditions for oxidative stress and inflammation assays.
Adenocarcinoma
Amelogenin
Amino Acids, Essential
Biological Assay
Caco-2 Cells
Cell Lines
Cells
Collagen
Colorectal Carcinoma
Enterocytes
Enzymes
Fetal Bovine Serum
Gender
Genetic Heterogeneity
Glutamine
Homo sapiens
Hyperostosis, Diffuse Idiopathic Skeletal
Inflammation
Membrane Transport Proteins
Microvilli
Oxidative Stress
Penicillins
Polyesters
Short Tandem Repeat
Streptomycin
Tight Junctions
Tissue, Membrane
Top products related to «Microvilli»
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A transmission electron microscope (TEM) is a type of electron microscope that uses a high-energy electron beam to image and analyze the internal structure of materials at the nanoscale level. The TEM functions by transmitting an electron beam through a thin specimen, allowing the beam to interact with the sample and create an image that can be magnified and projected onto a screen or captured digitally.
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More about "Microvilli"
Microvilli are the tiny, finger-like projections that extend from the surface of certain epithelial cells, such as those found in the intestinal lining.
These microscopic structures play a crucial role in increasing the surface area of the cell, allowing for more efficient absorption and transport of nutrients, ions, and other essential molecules.
Comprised of a core of actin filaments and covered by the cell membrane, microvilli are an important area of study in fields like cell biology, physiology, and digestive system disorders.
Researchers can leverage AI-driven protocol comparisons from tools like PubCompare.ai to optimize their microvilli research workflows, locate the best methods from literature, preprints, and patents, and improve reproducibility and accuracy.
Key techniques used in microvilli research include transmission electron microscopy (TEM), which can provide high-resolution images of these delicate structures.
Researchers may also utilize compounds like penicillin and streptomycin to maintain cell cultures, as well as specialized media like Leibovitz's L-15.
Data analysis software such as Prism and image processing tools like Embed 812 and CPD 030 can also support microvilli studies.
By understanding the structure, function, and regulation of microvilli, scientists can gain valuable insights into fundamental cellular processes and develop new treatments for digestive and other health conditions.
With the help of AI-driven research optimization, the field of microvilli biology continues to evolve and advance.
These microscopic structures play a crucial role in increasing the surface area of the cell, allowing for more efficient absorption and transport of nutrients, ions, and other essential molecules.
Comprised of a core of actin filaments and covered by the cell membrane, microvilli are an important area of study in fields like cell biology, physiology, and digestive system disorders.
Researchers can leverage AI-driven protocol comparisons from tools like PubCompare.ai to optimize their microvilli research workflows, locate the best methods from literature, preprints, and patents, and improve reproducibility and accuracy.
Key techniques used in microvilli research include transmission electron microscopy (TEM), which can provide high-resolution images of these delicate structures.
Researchers may also utilize compounds like penicillin and streptomycin to maintain cell cultures, as well as specialized media like Leibovitz's L-15.
Data analysis software such as Prism and image processing tools like Embed 812 and CPD 030 can also support microvilli studies.
By understanding the structure, function, and regulation of microvilli, scientists can gain valuable insights into fundamental cellular processes and develop new treatments for digestive and other health conditions.
With the help of AI-driven research optimization, the field of microvilli biology continues to evolve and advance.