Human type IV collagen (MilliporeSigma, St. Louis, MO) was deglucosylated in deglucosylation reaction buffer (50 mM acetate buffer pH5.3, 150 mM NaCl) at 37 °C for 4 h with 100 ug of PGGHG enzymes and 2500 µg type IV collagen. type IV collagen treated with inactive PGGHG D300E mutant served as a negative control. After incubation, the reaction was stopped by incubating at 98 °C for 3 min. The deglucosylated type IV collagen (dgCol4) production was indirectly detected by measuring glucose release using Glucose Colorimetric/Fluorometric Assay Kit (MilliporeSigma, St. Louis, MO) according to manufacturers’ instructions. Experiments were performed in duplicate from distinct samples, and an unpaired t-test was used to compare the enzymatic activity of different samples.
Collagen type 4
Manufactured by Merck Group
Sourced in United States, Switzerland, Belgium, Germany
Collagen type IV is a key structural component of basement membranes. It provides a platform for the assembly of other basement membrane proteins and plays a crucial role in the organization and function of this extracellular matrix.
Automatically generated - may contain errors
Lab products found in correlation
Fibronectin, by Merck Group (12 mentions)
Laminin, by Merck Group (11 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (5 mentions)
Lsm 700, by Zeiss (4 mentions)
Pneumacult ali medium, by STEMCELL (3 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (3 mentions)
Trypsin, by Thermo Fisher Scientific (3 mentions)
Bovine serum albumin (bsa), by Merck Group (3 mentions)
Collagen type 1, by Merck Group (3 mentions)
Efficient navigation zen , by Zeiss (2 mentions)
N n dimethylformamide (dmf), by Merck Group (2 mentions)
Bronchial epithelial growth medium (begm), by Lonza (2 mentions)
Axiovert 200m, by Zeiss (2 mentions)
Matrigel, by BD (2 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (2 mentions)
Flag tag (flag), by Merck Group (2 mentions)
Vitronectin, by Merck Group (2 mentions)
Fibronectin, by Abcam (2 mentions)
Gapdh, by Cell Signaling Technology (2 mentions)
Sodium chloride (nacl), by Merck Group (2 mentions)
90 protocols using collagen type 4
1
Deglucosylation of Type IV Collagen
2
Quantitative Cutaneous Nerve Fiber Analysis
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Biopsy specimens were fixed in Zamboni's solution (2% paraformaldehyde and picric acid) for two-dimensional assessments. The fixed samples were cryosectioned at thicknesses of 50 and 80 µm for IHC and IF, respectively. The first few and last few sections were discarded owing to potential artifacts.1 (link)2 (link) Three randomly selected sections of each thickness were used for subsequent immunostaining. The 50-µm-thick sections were immunolabeled with PGP 9.5 (1:2,000) using 3,3′-diaminobenzidine staining for IHC. The immunolabeled sections were imaged using bright-field microscopy (BX51, Olympus, Tokyo, Japan). The 80-µm-thick sections were immunostained with PGP 9.5 (1:800) and type IV collagen (1:800; Sigma-Aldrich, St. Louis, MO, USA) using indirect IF. We used confocal microscopy (LSM 700, Carl Zeiss, Jena, Germany) for image acquisition, and serial optical sections taken at 1-µm intervals were reconstructed using the related software (ZEN, Carl Zeiss). The maximum projection image derived from the stacked results was used to quantify cutaneous nerve fibers. The guidelines of the European Federation of Neurological Societies (EFNS) were adopted for quantification of the IENFD and the unit of results were represented as ‘IENFs/mm’.1 (link)2 (link)
3
Characterization of Primary Cell Cultures
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Masson stain Kit (Jiangcheng Corporation, China), trypsin and EDTA digestion solution (Solarbio Corporation, China), Dispase II (Roche Corporation, Switzerland), top grade fetal bovine serum (Sijiqing Corporation, China), K-SFM culture media (Gibco Corporation, USA), type IV collagen (Sigma Corporation, USA).
4
Progenitor hNEC Isolation and Expansion
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Progenitor hNECs were placed in DPBS with Ca2+/Mg2+ and stored at 4 °C until processing. They were removed from brushes and treated with a declumping solution containing PBS, dithiothreitol (3 mM), ethylenediaminetetraacetic acid (EDTA; 2 mM), and Type II collagenase (12.5 mg/100 ml) for 5–10 min at 37 °C followed by the addition of 10% FBS. Cells were plated on tissue culture dishes precoated with Type I bovine collagen (PureCol). They were expanded in bronchial epithelial cell growth media (BEGM) [35 (link)] or Ex Plus (Stem Cell Technologies) medium for 1 week before being cryopreserved. Upon thawing, the cells were plated on Type IV collagen (Sigma-Aldrich) coated Transwell inserts (Corning 3470; polyester, 6.5 mm diameter, 0.4 µm pores) and used as a control group.
5
Scratch Wound Healing Closure Assay
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The methods used for a scratch wound healing closure assay have been described previously [25 (link), 26 (link)]. Mouse HF keratinocytes isolated from K5-Cre;PDPNflox/flox and control mice (at postnatal day 4) were grown to full confluence in 25 μg/ml type IV collagen (collagen from human placenta, Sigma-Aldrich)-coated 24-well plates and were then incubated overnight in serum-reduced medium containing 1% FBS. Cells were scratched with a 200 μL sterile pipette tip and were then incubated. After 48 h, cells were washed with PBS and the images of the scratches were acquired. The surface areas of the cell-free zones were measured and the % scratch closure was determined using TScratch software [26 (link)].
6
Establishing PC-12 and Astrocyte Cell Cultures
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PC-12 cells were from the American Type Culture Collection (CRL-1721) and subjected to quality control tests by the American Type Culture Collection. PC-12 cells were cultured in Dulbecco's modified Eagle's medium (high glucose; Gibco) containing 10% heat-inactivated fetal bovine serum (FBS) (Gibco), 5% heat-inactivated horse serum (HyClone), penicillin (100 units/ml), and streptomycin (1 mg/ml) in plates coated with 10 μg/ml type IV collagen (Sigma–Aldrich). Cells were passaged no more than eight times.
Astrocytes were isolated from Sprague–Dawley rat pup brains, as previously described (67 (link)). In brief, cortices were dissected from the forebrain and surrounding meninges and then mechanically and enzymatically dissociated using the Neural Tissue Dissociation Kit P (Miltenyi Biotec). Mixed glial cultures were established in Dulbecco's modified Eagle's medium/F-12 medium supplemented with GlutaMAX (Gibco), 10% FBS, and 100 units/ml antibiotic–antimycotic (Gibco). After culturing for 10 to 14 days, microglia and oligodendrocytes were removed by shaking. The astrocytes were collected by trypsinization and replated at 3.5 × 105 cells/well on poly-d -lysine-coated surfaces. Experiments were performed within 48 h of completing the isolation procedure.
Astrocytes were isolated from Sprague–Dawley rat pup brains, as previously described (67 (link)). In brief, cortices were dissected from the forebrain and surrounding meninges and then mechanically and enzymatically dissociated using the Neural Tissue Dissociation Kit P (Miltenyi Biotec). Mixed glial cultures were established in Dulbecco's modified Eagle's medium/F-12 medium supplemented with GlutaMAX (Gibco), 10% FBS, and 100 units/ml antibiotic–antimycotic (Gibco). After culturing for 10 to 14 days, microglia and oligodendrocytes were removed by shaking. The astrocytes were collected by trypsinization and replated at 3.5 × 105 cells/well on poly-
7
Schwann Cells Migration Assay
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SCs migration assays were performed using a BD BioCoat cell culture insert with a 6.5 mm diameter filter and 8 µm pore size following the manufacturer’s instructions (BD Biosciences). First, laminin (2 μg/mL), fibronectin (2 μg/mL), type IV collagen (5 μg/mL) (all from Sigma), and PBS (1x) were preplated on polycarbonate membranes (membrane between the upper and lower chambers to which SCs could attach) and then SCs were seeded (1.5 × 105 cells/insert) into the upper chamber containing complete medium supplemented with 2 mM L-glutamine, 50 U/mL penicillin, 50 mg/L streptomycin and 1% DMEM high glucose medium (v/v). Complete medium containing UTP was placed in the lower chamber as a chemoattractant. After 24 h of incubation in a humidified environment with 5% CO2 at 37°C, cells that migrated to the lower chamber were fixed with 70% (v/v) ethanol and stained with 0.2% (w/v) crystal violet. For each polycarbonate membrane, the number of cells was counted under a light microscope in 4 randomly selected random fields. The number of migrated cells in the Control group (PBS) was used as a reference value of 1.
8
Extracellular Matrix Protein Preparation
9
Airway Epithelium Differentiation Assay
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BC differentiation was assessed using air–liquid interface (ALI) cultures. After the BC reached 70–80% confluence, the cells were trypsinized and seeded (density 1.5 × 105 cells/cm2) onto a 0.4 μm pore-sized Costar Transwells inserts (Corning) pre-coated with type IV collagen (Sigma) in PneumaCult™-Ex Plus Medium. After confluence, the basolateral medium was replaced with PneumaCult™-ALI Medium (StemCell Technologies), and the apical surface exposed to air (“ALI day 0”). The medium was changed every other day until ALI day 28, a time-point when normal BC generate a fully differentiated mucociliary airway epithelium. Epithelial barrier integrity was assessed by measuring Rt (Millicell-ERS epithelial ohmmeter, Millipore). At day 28, ALI culture inserts were washed with PBS for 2 times and fixed in 4% paraformaldehyde at room temperature for 30 min. After two PBS washes, fixed inserts in 70% ethanol Inc for low-melt paraffin embedding and sectioning then washed with PBS twice and embedded in low-melt paraffin block (Histoserv Inc, Germantown, MD). Five micrometer sections were cut for each ALI culture and stained with hematoxylin and eosin for histological analysis.
10
Isolation of Highly-Adherent Cells from Differentiating hiPSCs
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It was found necessary to isolate a high-adherence population of cells from the differentiating cells, as unfractionated cells could detach from the hydrogel-substrate, leaving gaps in the monolayers, resulting in unusable cultures. On day 8 after shift to UC medium, the cells were detached with accutase (45 min, 37°C) and resuspended in EC medium. They were seeded onto collagen- and fibronectin-coated 6-well plates (Nunc) prepared one day in advance. These adherence plates were coated with 1ml per well of HBSS containing 16μg/ml type-IV collagen (Sigma-Aldrich) and 4μg/ml fibronectin (Santa Cruz). One adhesion plate was used for each plate of differentiated hiPSCs.
The cells were incubated for 60 min at 37°C on the adhesion plates and weakly-adherent cells were removed by aspiration followed by two washes with HBSS at 37°C. Strongly adherent cells were released with accutase, and these cells were used for further seeding onto hydrogels or transwell inserts.
The cells were incubated for 60 min at 37°C on the adhesion plates and weakly-adherent cells were removed by aspiration followed by two washes with HBSS at 37°C. Strongly adherent cells were released with accutase, and these cells were used for further seeding onto hydrogels or transwell inserts.
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