Collagen 1 from rat tail

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Collagen I from rat tail is a naturally derived extracellular matrix protein. It is obtained from the tendons of rat tails. Collagen I is a fibrillar collagen that provides structural support and promotes cell attachment and proliferation in cell culture applications.

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Collagen I (143 citations) Rat tail collagen I (115 citations) Rat tail collagen (30 citations) Rat collagen I (5 citations) Collagen from rat tail (type I), (2 citations)

11 protocols using collagen 1 from rat tail

Aminoglycoside Aptamer Purification and Characterization

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Tris-2-carboxyehyl-phosphine (TCEP), 6-mercapto-1-hexanol (99%), Trizma base (2-amino-2-(hydroxymethyl)-1,3-propanediol, magnesium chloride (MgCl₂), sodium chloride (NaCl), tobramycin (Tob), ferrocene carboxylic acid, 97% (FCC), sulfuric acid (H₂SO₄), and 10X Tris-EDTA buffer, Dulbecco’s modified Eagle’s medium (DMEM), sodium hydroxide (NaOH), sodium acetate (NaOAC) Tetrabutylammonium hexafluorophosphate (TBAPF₆), ferrocene (FC) Fetal Bovine Serum (FBS), and Protector RNase Inhibitor were all used as received from Sigma-Aldrich. Hydrogen peroxide 30%, 95% ethanol, and 10x PBS buffer were used as received (Fischer Scientific). Collagen I from rat tail was used as received (Gibco). Ambion RNaseAlert QC System was used as obtained from Thermo Fischer Scientific. SP Sepharose Fast Flow was used as received from GE Healthcare Life Sciences. All solutions were prepared using autoclaved, ultrapure water (18.0 MΩ cm at 25 °C) using a Biopak Polisher Millipore ultra-purification system (Millipore, Billerica, MA). The RNA aminoglycoside aptamer sequence (5′-HSC6-CUUGGUUUAGGUAAUGAG-MB-3′ (D2 Sequence)^{16 (link)} was purified using dual-HPLC (Biosearch Technologies, CA) and used as received.

Santos-Cancel M, & White R.J. (2017). Collagen Membranes with Ribonuclease Inhibitors for Long-Term Stability of Electrochemical, Aptamer-Based Sensors Employing RNA. Analytical chemistry, 89(10), 5598-5604.

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Detailed Protocol for Detroit 562 Pharyngeal Cell Monolayer Formation

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Detroit 562 pharyngeal cell monolayers form the substratum for GAS biofilm growth. An outline of the process and the monolayers formed is depicted in Fig. 1.

The wells of a 96-well flat bottom cell culture microtiter plate (Greiner Bio-One, Germany) were coated with 50 µL of 300 µg/mL collagen I from rat tail (Gibco, Life Technologies, UK) prepared in pre-chilled, sterile 17.4 mM acetic acid solution. The plate was incubated for 1 h, 37 °C, 5% CO₂–20% O₂ atmosphere.

After 1 h, excess collagen was removed, and the wells seeded with 150 µL Detroit 562 cell suspension (2 × 10⁵ cells/mL) and cultured for 48 h (to achieve ~ 95% confluency).

Monolayers were washed once with 200 µL of sterile PBS, and fixed with 50 µL sterile 3.7% paraformaldehyde (PFA) (w/v) for 20 min.

Once cells were fixed, PFA was removed and wells washed twice with 200 µL of PBS.

Monolayers can be used immediately, or stored at 2–8 °C for up to 2 weeks (with monolayers kept wet via submersion in 200 µL of sterile PBS) until required for use.

Figure 1

Schematic outlining the process of Detroit 562 pharyngeal cell monolayer formation. Schematic shows collagen coating, seeding with Detroit 562 pharyngeal cells, and finally an example well containing a 3.7% PFA fixed ~ 95% confluent monolayer of Detroit 562 pharyngeal cells. Example monolayer image taken at ×10 objective in an Incucyte^® S3 Live-Cell Analysis System.

Vyas H.K., McArthur J.D, & Sanderson-Smith M.L. (2021). An optimised GAS-pharyngeal cell biofilm model. Scientific Reports, 11, 8200.

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Neuronal Cell Culture Procedure

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Fetal bovine serum (FBS), 7.5% sodium bicarbonate solution (NaHCO₃), cell culture grade water, 10,000 U/mL Penicillin/Streptomycin (Pen/Strep), 10× Dulbecco’s Modified Eagle’s Medium- low glucose (DMEM), PC12 cells, and ionomycin from Streptomyces conglobatus were used as acquired from Sigma. Sodium L-glutamate monohydrate and 1,2-Bis(2-aminophenoxy)ethane-N,N,N’, N’-tetraacetic acid tetrakis(acetoxymethyl ester) (BAPTA-AM), Heat-Inactivated (HI) Horse Serum were acquired and used as received from Thermo Fisher. Rat primary cortical astrocytes were received from Invitrogen. Dulbecco’s Modified Eagle Medium (DMEM) (+4.00 mM L-Glutamine, +450 mg/L glucose, -sodium pyruvate) and 10× PBS buffer (Hyclone); trypan blue (0.4%), collagen I from rat tail, and 0.25% trypsin (1×) (Gibco); Live/Dead Cell Imagining Kit for mammalian cells (Invitrogen) were used as received. RPMI-1640 (high glucose, with L-glutamine, with HEPES) was obtained from ATCC.

Santos-Cancel M., Simpson L.W., Leach J.B, & White R.J. (2019). Direct, Real-Time Detection of Adenosine Triphosphate Release from Astrocytes in 3-Dimensional Culture Using an Integrated Electrochemical, Aptamer-Based Sensor. ACS chemical neuroscience, 10(4), 2070-2079.

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Lysosomal Dynamics in Cell Culture

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RPMI 1640 medium (RPMI), fetal bovine serum (FBS), collagen I from rat tail, and Penicillin/Streptomycin were purchased from Gibco (Grand Island, NE, USA). Fluoromount G and DAPI were obtained from Life Technologies (Carlsbad, CA, USA). LysoTracker^® DND-99 probe, Alexa Fluor^® 647 Phalloidin, and Actin Red™555 ReadyProbes™ Rhodamine phalloidin were commercially obtained (Thermo Scientific, Waltham, MA, USA). Murine Interferon Gamma (IFN-γ) was purchased from Prepotech. [4, 5-dimethyl-thiazol-2-yl]-2, 5-diphenyltetrazolium bromide (MTT), neutral red solution (NR), and dimethyl sulfoxide (DMSO) were obtained from Sigma-Aldrich (St. Louis, MO, USA). CQ was purchased from Cristália (São Paulo, Brazil). Western Blotting reagents were obtained from GE HealthCare Life Sciences (Little Chalfont, UK). All other reagents were obtained from Sigma-Aldrich (St. Louis, MO, USA) if not otherwise specified.

Bosquetti B., Santana A.A., Gregório P.C., da Cunha R.S., Miniskiskosky G., Budag J., Franco C.R., Ramos E.A., Barreto F.C, & Stinghen A.E. (2023). The Role of α3β1 Integrin Modulation on Fabry Disease Podocyte Injury and Kidney Impairment. Toxins, 15(12), 700.

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Engineered Collagen Tissue Construct

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Collagen I from rat tail (Gibco), populated with fibroblasts that were isolated from foreskins, was used for fabricating successive layers of acellular and cellular collagen on a polycarbonate membrane. The collagen I was prepared as previously described44 (link). After approximately 7 days, 1 × 10⁵ hAFS-K cells were added to the centre of the collagen gel and incubated for 1 h at 37 °C to allow the hAFS-K cells to fully adhere. Cells were exposed to the air-liquid interface for 8 days in Epilife medium (Gibco).

Sun Q., Li F., Li H., Chen R.H., Gu Y.Z., Chen Y., Liang H.S., You X.R., Ding S.S., Gao L., Wang Y.L., Qin M.D, & Zhang X.G. (2015). Amniotic fluid stem cells provide considerable advantages in epidermal regeneration: B7H4 creates a moderate inflammation microenvironment to promote wound repair. Scientific Reports, 5, 11560.

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Culturing and Fixing Detroit 562 Cells

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Detroit 562, a human pharyngeal epithelial cell line (CellBank Australia, Westmead, Australia), was cultured in Dulbecco’s Modified Eagle Medium (DMEM) F12 (Gibco, Grand Island, NY, USA), supplemented with 2 mM L-glutamine (Gibco, Life Technologies, Grand Island, NY, USA) and 10% (v/v) heat inactivated foetal bovine serum (FBS) (Bovogen Biologicals, Keilor East, Australia) in cell culture flasks at 37 °C, 5% CO₂ to 20% O₂ atmosphere.
Fixed Detroit 562 pharyngeal cell monolayers form the substratum for bacterial growth for subsequent biofilm experiments. In brief, wells of 96-well flat bottom cell culture microtiter plates (Greiner Bio-One, Frickenhausen, Germany) were coated with 300 µg/mL Collagen I from rat tail (Gibco, Life Technologies, Grand Island, NY, USA) and incubated for 1 h, 37 °C, 5% CO₂ to 20% O₂ atmosphere. After 1 h, wells were seeded with 150 µL Detroit 562 cell suspension (2 × 10⁵ cells/mL) and cultured for 48 h (or until a monolayer of ~95% confluency was achieved). Monolayers were washed once with PBS and fixed with sterile 3.7% paraformaldehyde for 20 min. Once fixed, wells were washed twice with PBS, and monolayers were kept wet via submersion in PBS until required for use.

Vyas H.K., Indraratna A.D., Everest-Dass A., Packer N.H., De Oliveira D.M., Ranson M., McArthur J.D, & Sanderson-Smith M.L. (2020). Assessing the Role of Pharyngeal Cell Surface Glycans in Group A Streptococcus Biofilm Formation. Antibiotics, 9(11), 775.

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Visualizing Histone Dynamics in U2OS Cells

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U2OS (Human Osteosarcoma) cells were grown in DMEM (Life Technologies) with 1 g/l glucose and glutamax supplemented with 10% FBS (Fetal Bovine Serum, Life Technologies) and 1% Penicillin/Streptomycin (Life Technologies) at 37 °C with 5% CO2. Forty-eight hours prior to fixation, cells were seeded at 30–40% confluence on plasma-cleaned (2 mn with air with Femto model, Diener Electronic) and collagen-coated (Collagen I from Rat tail, Life Technologies) coverslips (N°1 25 mm).
Plasmid H2B-pDendra2(N) was obtained by cloning H2B coding sequence into pDendra2-N vector (evrogen). Cells were transfected 24 h before imaging with the H2B-pDendra2(N) plasmid (100 ng/25 mm coverslip) using Fugene 6 (Roche Applied Science, IN46250-0414) according to manufacturer instructions. Fixation was performed using para-formaldeïd (4%) (Electron Microscopy Sciences #15714) for 15 min before washing and imaging in PBS.

Récamier V., Izeddin I., Bosanac L., Dahan M., Proux F, & Darzacq X. (2014). Single cell correlation fractal dimension of chromatin: A framework to interpret 3D single molecule super-resolution. Nucleus, 5(1), 75-84.

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3D Collagen Scaffold Formation

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3D scaffolds were generated with Collagen I from rat tail (Life Technologies, Carlsbad, CA, USA). Gelation was performed according to the manufacturer recommendations as previously described [11 (link)]. Collagen was diluted at 2 mg/mL in PBS (Phosphate Buffer Saline, Life Technologies, Carlsbad, CA, USA) and pH at 7.0 was obtained using sterile NaOH (Merck Millipore, Burlington, MA, USA). Collagen is then incubated at 37 °C in a humidified incubator for 40 min. Gels were gently rinsed with PBS for 15 min before cell seeding.

, & Millet A. (2021). A Universal Model for the Log-Normal Distribution of Elasticity in Polymeric Gels and Its Relevance to Mechanical Signature of Biological Tissues. Biology, 10(1), 64.

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Embryonic Heart AVC Explant Culture

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The AVC region of the heart at the E9.5 embryo stage was dissected out and opened with fine iris scissors under a dissecting microscope. The endothelial surface was placed toward collagen gel containing 1 mg/mL collagen I from rat tail (Thermo Fisher Scientific) in Medium 199. The explant was allowed to attach at 37°C in a humidified atmosphere of 5% CO₂ overnight and was cultured in 0.1 mL of Medium 199 supplemented with 1% FBS, 0.01% Insulin-Transferrin-Selenium (Thermo Fisher Scientific), and penicillin/streptomycin at 37°C in a humidified atmosphere of 5% CO₂ for 48 h [15 (link)]. Transformed cells showing stellate and spindle-shapes were counted under an inverted light microscope (Axiovert 40C,) as described previously [15 (link)–17 (link)]. PI3K inhibitor LY294002 (Merck KGaA) and MEK inhibitor PD0325901 (Selleckchem) were dissolved in DMSO, and an equal amount of stock solution was added to the culture media (final DMSO concentration, 0.2%).

Kim K, & Lee D. (2021). ERBB3-dependent AKT and ERK pathways are essential for atrioventricular cushion development in mouse embryos. PLoS ONE, 16(10), e0259426.

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Monocyte Isolation and Macrophage Differentiation

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Monocytes were isolated from the bone marrow of 7 to 10 week-old C57BL/6 male mice (Jackson Laboratory, Bar Harbor, ME) as described previously^{32 (link)}. Cells were separated using Lympholyte M (Accurate Chemical, Westbury, NY) and plated in macrophage differentiation medium (IMDM with 20% FBS, 2 mM L-glutamine (Thermo), 1% penicillin-streptomycin (Corning, Corning, NY), 1.5 ng/ml recombinant mouse macrophage colony stimulating factor (M-CSF, R&D Systems, Minneapolis, MN), and 100 ng/ml flt-3 ligand (R&D Systems) for 5 days. Macrophages were lifted from culture plates using a cell scraper and 0.05% trypsin-EDTA. A soybean trypsin inhibitor (Thermo) was used in place of serum. Cells were seeded at 30,000 cells/cm² in serum-free medium on hydrogels swollen in PBS or in 10% FBS in PBS, Human Plasma Fibronectin (Millipore, Billerica, MA), Collagen I from rat tail (Thermo), or active mouse Fibrinogen protein (ab92791, Abcam) for 30 minutes prior to seeding. After 24 hours, hydrogels were rinsed, fixed in 4% formaldehyde, and adhered macrophages were stained with DAPI at 1:10000 (Thermo). Adhered macrophages were imaged on a Zeiss Axio Observer Z1 microscope (Carl Zeiss, Oberkochen, Germany) using an AxioCam MRm camera and an EC Plan-Neofluar 20X 0.4 NA air objective and manually quantified using ImageJ (NIH, Bethesda, MD).

Jansen L.E., Amer L.D., Chen E.Y., Nguyen T.V., Saleh L.S., Emrick T., Liu W.F., Bryant S.J, & Peyton S.R. (2018). Zwitterionic PEG-PC hydrogels modulate the foreign body response in a modulus-dependent manner. Biomacromolecules, 19(7), 2880-2888.

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