All animal studies were carried out with prior approval by the Institutional Animal Care and Use Committee of the University at Buffalo. The procedures for preparing rat cochlear organotypic cultures have been described previously (Ding, et al, 2002 (link), 2011 (link), 2012 (link), 2014 (link)). Briefly, on postnatal day 3, rat pups were decapitated. The cochleae were then removed and placed in Hank’s balanced salt solution (1X GIBCO, 14175, Invitrogen, Carlsbad, CA). After removing the lateral wall, the whole basilar membrane containing the organ of Corti and spiral ganglion neurons were isolated, transferred onto a drop of 10 μl collagen gel matrix in a 35 mm culture dish. The collagen gel was prepared with a solution consisting of 15 μl of rat tail collagen (Type 1, BD Biosciences, 4236 Bedford, MA), 10×basal medium eagle (BME, Sigma B9638), and 2% sodium carbonate in a 9:1:1 ratio. The collagen gel was submerged in 1.3 ml of serum-free medium consisting of 2 g bovine serum albumin (BSA, Sigma A-4919), 2 ml serum-free supplement (Sigma I-1884), 4.8 ml of 20% glucose (Sigma G-2020), 0.4 ml penicillin G (Sigma P-3414), 2 ml of 200 mM glutamine (Sigma G-6392), and 190.8 ml of 1X BME (Sigma B-1522), and the cochlear explants were then incubated for 24 h (Forma Scientific 3029, 37°C in 5% CO2).
Rat tail collagen type 1
Manufactured by BD
Sourced in United States, United Kingdom, Germany, Morocco, Italy, Belgium
Rat tail collagen type I is a natural extracellular matrix component derived from the tails of rats. It is a fibrillar collagen that provides structural support and promotes cell attachment and proliferation in various cell culture and tissue engineering applications.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (22 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (10 mentions)
Matrigel, by BD (9 mentions)
Bovine serum albumin (bsa), by Merck Group (8 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (8 mentions)
Penicillin, by Thermo Fisher Scientific (7 mentions)
Streptomycin, by Thermo Fisher Scientific (7 mentions)
Egm 2, by Lonza (7 mentions)
Ebm 2 medium, by Lonza (6 mentions)
Acetic acid, by Merck Group (5 mentions)
Hepes, by Merck Group (5 mentions)
L glutamine, by Thermo Fisher Scientific (5 mentions)
α mem, by Thermo Fisher Scientific (4 mentions)
Human plasma fibronectin, by Merck Group (4 mentions)
Pen strep, by Thermo Fisher Scientific (4 mentions)
Trypsin edta, by Thermo Fisher Scientific (4 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (4 mentions)
Fetal bovine serum (fbs), by Merck Group (3 mentions)
Antibiotic antimycotic, by Thermo Fisher Scientific (3 mentions)
Egm 2 bulletkit, by Lonza (3 mentions)
257 protocols using rat tail collagen type 1
1
Rat Cochlear Organotypic Culture Protocol
2
Collagen Matrix Preparation for Cell Culture
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The collagen matrix was made as previously described [8 (link)] by mixing on ice: 444 μL of rat tail collagen type 1 (3.40 mg/mL, BD Biosciences, MA-USA), 64 μL of 10X DMEM, 128 μL of reconstitution buffer (RB) pH 8.15 [2.2 g NaHCO3, 0.6 g NaOH, and 4.766 g 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid in 100 mL dH2O], and 64 μL of FBS per well of 24-well culture plate (BD Falcon, NJ-USA). The pH was adjusted to 7.2–7.4 by using RB. Seven hundred μL of this collagen matrix mixture was layered uniformly in each well of 24-well culture plate. The matrix was allowed to polymerize for 1h in a humidified chamber at 37°C and then equilibrated with 1 mL of routine fibroblast culture medium.
3
Adipose-Derived Stem Cell Culture
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Cells. Human adipose derived stem cells in passage 2 were seeded in at 112 500cells/cm2 on inserts for 24-well plates with aligned or random PCL-fibers or at 56 250 cells/cm2 on glass cover slides (170 µm thickness) coated with rat tail collagen type 1 (BD Bioscience, Bedford, MA, USA) and differentiated as described above. The cells were fixed on indicated time points using PFA and stored in PBS until analysis.
4
Three-Dimensional Cell Invasion Assay
5
Rat Hepatocyte Isolation and Culture
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HepG2 cell line was maintained in modified essential medium (MEM) supplemented with 10% fetal bovine serum (FBS) plus sodium pyruvate and antibiotics (penicillin/streptomycin). Hepatocytes were isolated from livers of male Wistar rats approximately 200–250 g of weight. Animals were maintained on an ad libitum diet and handled according to internal institutional guidelines and ethical agreements (Instituto de Fisiología Celular, Universidad Nacional Autónoma de México) for animal experimentation. Rat hepatocytes were isolated using the modified collagenase perfusion method from Berry and Friend as previously described in Caligaris et al.34 (link) After digestion, hepatocytes were separated by centrifugation at 400 r/min for 2 min from non-parenchymal cells, and viable hepatocytes were isolated by iso-density percoll centrifugation (Amersham-GE Life Science). Cell viability was evaluated by trypan blue exclusion staining. For primary culture, hepatocytes were seeded on coverslips coated with PDMS and treated with 1 mg/mL rat tail collagen type 1 (BD Biosciences), and cells were cultured for 4 h at 37°C in attachment medium supplemented with 10% FBS and antibiotics. Then, media was changed for feeding medium (FBS-free) and hepatocytes were cultured for 24 h for further studies.
6
Cell Culture and Differentiation of Airway Epithelial Cells
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HBEC3kt cells were cultured in keratinocyte serum-free medium (KSFM; Thermo, Fisher Scientific, Grand Island, NY) supplemented with human epidermal growth factor and bovine pituitary extract. MLE-15 cells were cultured in RPMI supplemented with 10% fetal bovine serum. Cultures were maintained in the presence of penicillin and streptomycin. NHBE cells were expanded in submerged culture using a Clonetics B-ALI air-liquid interface protocol and reagents (Lonza) and then seeded into 24-well plates containing transwell inserts coated with rat tail collagen type 1 (BD Biosciences, East Rutherford, NJ). Three days after seeding, B-ALI growth medium was removed from both apical and basal chambers, and B-ALI differentiation medium was added to only the basal chambers.
7
Isolation and Labeling of ECFCs
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Fresh human cord blood, donated under full ethical approval by healthy volunteers at the Northern Ireland Blood Transfusion Service (Belfast, U.K.), underwent density gradient fractionation for the isolation of mononuclear cells and was selected for ECFCs via resuspension in complete medium (EBM-2 plus EGM-2 MV supplements, Lonza Group) supplemented with 10% FBS and seeding onto 24-well culture plates precoated with rat tail collagen type 1 (BD Biosciences, Bedford, U.K.) at a density of 1 × 107 cells/mL. Cells were labeled (Qtracker 655, Invitrogen, Life Technologies, Carlsbad, CA) per the manufacturer’s instructions.
8
Isolation and Culturing of BOECs
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BOECs were isolated as previously described with some modifications [24 (link)]. Peripheral blood samples were collected from healthy adult Europeans (30 mL; n = 5) and Ghanaian children (2–8 mL; n = 34) in lithium heparin tubes (Becton Dickson). Blood samples were diluted 1:1 with PBS, and peripheral blood mononuclear cells (PBMCs) isolated by density-gradient centrifugation over Histopaque (GE) or Lymphoprep (StemCell Technologies). European PBMCs were washed twice with PBS, then seeded in six-well culture plates (Corning) coated with 50 μg/mL rat tail collagen type 1 (BD Biosciences). Medium was changed every second day once colonies (from day 6 post-seeding) started to appear. Following isolation, PBMCs from the Ghanaian children were washed twice with PBS, and suspended in 350 μL EGM-2 plus Bullet Kit (Lonza) supplemented with 10% FBS, 50 μL DMSO, and 100 μl FBS. PBMC from the Ghanaian children were frozen using a Mr. FrostyTM freezing container and stored overnight at -80˚C, then transferred to liquid nitrogen. Following transfer to Denmark, the frozen PBMC were thawed, washed in pre-warmed EGM-2 and processed as above. All experiments described in this paper were carried out with BOECs at passage 2 to 8.
9
Chemotaxis Assay for SGHPL-4 Cells
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SGHPL-4 cell chemotaxis toward rDSC CM was measured using the μ-Slide Chemotaxis 2D assay (Ibidi, Martinsried, Germany), according to the manufacturer’s instructions and a previously described method (Wallace et al. 2013 (link)).
In brief, cultivation areas of µ-slide were coated in 60 µg/mL rat tail collagen Type 1 (BD Biosciences) in 0.02 M acetic acid. Each chamber on the µ-slide was seeded with 1.8 × 104 cells and incubated for approximately 4 h to allow cells to adhere. rDSC or DSC control medium was pipetted into the upper and lower reservoirs of each chamber and DSC CM or rDSC CM applied to either the upper or lower reservoir.
Analysis of chemotaxis was performed by time-lapse microscopy using an Olympus IX70 inverted microscope, as described earlier. Images were taken every 15 min for 24 h, and time-lapse sequences were analysed using ImageJ software, version 1.46r with the Manual Tracking and Chemotaxis tool, version 1.01 plug-ins. Analysis of directional cell movement was measured by randomly choosing 20 cells in two fields of view, which were manually tracked over 24 h. The number of cells moving towards a chemotactic stimuli were recorded.
In brief, cultivation areas of µ-slide were coated in 60 µg/mL rat tail collagen Type 1 (BD Biosciences) in 0.02 M acetic acid. Each chamber on the µ-slide was seeded with 1.8 × 104 cells and incubated for approximately 4 h to allow cells to adhere. rDSC or DSC control medium was pipetted into the upper and lower reservoirs of each chamber and DSC CM or rDSC CM applied to either the upper or lower reservoir.
Analysis of chemotaxis was performed by time-lapse microscopy using an Olympus IX70 inverted microscope, as described earlier. Images were taken every 15 min for 24 h, and time-lapse sequences were analysed using ImageJ software, version 1.46r with the Manual Tracking and Chemotaxis tool, version 1.01 plug-ins. Analysis of directional cell movement was measured by randomly choosing 20 cells in two fields of view, which were manually tracked over 24 h. The number of cells moving towards a chemotactic stimuli were recorded.
10
Collagen Gel Organotypic Skin Model
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Collagen gels containing J2 fibroblast feeder cells were prepared from a mix of rat tail collagen type 1 (BD Biosciences), 10× reconstitution buffer (2.2 g NaHCO3, 4.8 g HEPES in 100 ml 0.05 M NaOH), and 10× Dulbecco’s modified Eagle’s medium (DMEM) without NaHCO3. Gels were allowed to solidify in a 6-well cell culture dish for at least 1 h. Between 1 × 106 and 2 × 106 control or knockdown cells were seeded onto the top of a collagen gel and grown to confluence in E-medium with EGF. At confluence, E-medium was removed, and the collagen gel was transferred onto a metal grid in a 100-mm dish. An air-liquid interface was created by adding E-medium without EGF to the bottom of the dish so that it touches the metal grid but not the collagen. Rafts were incubated at 37°C, and medium was changed every other day. Rafts were harvested after 14 days and fixed, and paraffin blocks were generated by the Skin Disease Research Center at Northwestern University. This was followed by hematoxylin and eosin (H&E) staining for analysis.
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