Organotypic culture (OTC) were grown as previously described [14 (link)] with the exception that for 2 more days before the cultures were harvested medium was changed to serum free Epi3 to allow for analysis of the conditioned media. To alter the matrix composition to higher collagen I, we used collagen I (BD Biosciences, San Jose, CA) at the final concentration of 4.6 mg/mL either mixed with Matrigel or applied as collagen I layer on top of the matrix. Conditioned media were snap frozen and conserved at −80C. Each cultures was used for protein analysis by formalin fixation and paraffin embedding, as well as protein extraction by mechanically peeling the epithelium. The protein were extracted with lysis buffer containing NP40 and Tween20 as previously described [12 ]. Similarly, total RNA from the epithelium and stroma was isolated using miRNeasy kit (Qiagen). Quantification of invasion was done by measuring the area of epithelial cells invading in the underlying matrix per 1µm length of tissue and statistically analyzed using unpaired Student’s t-test.
Collagen 1
Manufactured by BD
Sourced in United States, United Kingdom, France, Germany, Italy
Collagen I is a type of extracellular matrix protein that serves as a structural component in various tissues, including skin, bone, and tendon. It provides a scaffold for cell attachment and supports the integrity of these tissues.
Automatically generated - may contain errors
Lab products found in correlation
Matrigel, by BD (25 mentions)
Fibronectin, by BD (11 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (8 mentions)
Fibronectin, by Merck Group (8 mentions)
Laminin, by BD (6 mentions)
Laminin, by Merck Group (4 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (4 mentions)
Bovine serum albumin (bsa), by Merck Group (4 mentions)
Collagen 4, by Merck Group (4 mentions)
Dmem f12, by Thermo Fisher Scientific (4 mentions)
Insulin, by Merck Group (4 mentions)
Dulbecco modified eagle medium (dmem), by Merck Group (3 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (3 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (3 mentions)
Glutamax, by Thermo Fisher Scientific (3 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (3 mentions)
Gelatin, by Merck Group (3 mentions)
Collagen 4, by BD (3 mentions)
Matrigel, by Thermo Fisher Scientific (3 mentions)
Collagen 1, by Thermo Fisher Scientific (3 mentions)
161 protocols using collagen 1
1
Organotypic Culture Analysis of Epithelial Invasion
2
LPA Receptor Expression and Signaling
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Fetal bovine serum (FBS) was obtained from Nichirei Biosciences Inc. (Tokyo, Japan); RPMI1640 and D-MEM were from Sigma-Aldrich (St. Louis, MO, USA); Collagen I, Matrigel (growth factor reduced), fibronectin, laminin, and Collagen I pre-coated coverslips were obtained from Becton Dickinson (San Jose, CA, USA); fatty-acid-free bovine serum albumin (BSA) from Nacalai Tesque (Kyoto, Japan); LPA from Enzo Life Sciences Inc. (Farmingdale, NY, USA); Opti-MEM from Thermo Fisher Scientific Inc. (Waltham, MA, USA); Anti- LPA1, LPA3, LPA5, and LPA6 rabbit polyclonal antibodies from GeneTex Inc. (Irvine, CA, USA); anti-LPA 2 rabbit polyclonal antibody from Abgent (San Diego, CA, USA); and anti-LPA4 rabbit polyclonal antibody from Acris Antibodies Inc. (San Diego, CA, USA); Ki16425 was purchased from Cayman Chemical (Ann Arbor, MI, USA).
3
3D Collagen Gel Cell Culture Assay
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A 1.6 mg/mL collagen I solution was prepared by mixing rat tail collagen I (BD Biosciences) with deionized water, 10X PBS (Sigma, 1:10 dilution) and 1 N NaOH (Sigma, 1:1000 dilution) on ice. Collagen solution (150 μL) was placed in each well of a 48-well plate. Gels were incubated at 37 °C for 1 hour, after which 1.875E5 231BR cells in 400 μL of RPMI containing 10% FBS were seeded onto each gel. Control gels received 400 μL of RPMI containing 10% FBS. After incubation at 37 °C for 3 days, cells were extracted from the gels. The media was removed from each well and the gels were washed 3 times with 1X PBS (Life Technologies). A 20mM NH4OH solution containing 0.5% (v/v) Triton X-100 (Sigma) was added to each well and the chamber slide was incubated at 37 °C for 5 minutes. After removal of the NH4OH/Triton X-100 solution, the gels were washed twice with distilled water and twice with PBS. 175 μL of PBS was added to each well prior to imaging.
4
3D Collagen Gel Cell Culture Assay
5
Transfection of Mouse Podocytes
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Transient transfections were carried out in immortalized mouse podocyte cells cultured in RPMI with glutamax (Invitrogen) and supplemented with 10% FBS (Invitrogen Corp.) and 200 U/ml penicillin and streptomycin (Roche Applied Science) along with ITS (Insulin, Transferrin and Selenium) (Invitrogen). Transfections were performed using Lipofectamine 2000 (Invitrogen Corp.), Fugene HD (Roche) and electroporation using Amaxa nucleofactor II (Amaxa biosystem) as per manufacturer’s directions. Collagen 1 (4.73 mg/ml, #354236) or fibronectin (1 mg/ml diluted in PBS, #356008) were obtained from BD Biosciences, Bedford, MA. Laminin (0.5 mg/ml in TBS, #L6274) was obtained from Sigma-Aldrich; St. Louis, MO. 2–3 ml of Laminin, Collagen 1 and fibronectin was added to 6-well culture plates to completely coat the well- surface and incubated for 2 h at 37°C. Plates were then washed thoroughly with PBS prior to plating of cells in complete media.
6
Traction Force Microscopy of Cell Pairs
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Traction force microscopy was performed as previously described (Sabass et al., 2008 (link); Oakes et al., 2012 (link)). In brief, PA gels with a Young’s modulus of 8.4 kPa were polymerized with 80-nm fluorescent microspheres (Invitrogen) on prepared glass coverslips and covalently conjugated with collagen 1 (BD) using sulphosanpah (Thermo Fisher Scientific). Cells were plated on gels and allowed to spread overnight. As cells readily formed pairs on PA gels, cell pairs were analyzed rather than single cells. Images of microspheres were captured before and after cell removal with a 0.5% SDS solution, and image pairs were registered and analyzed by PIV as in the previous section. Traction stresses were calculated from the displacement field by Fourier transform traction cytometry using zeroth-order regularization.
7
Sterilization and Surface Coating of Microfluidic Device
8
Differentiation and Culture of PC12 and HT-22 Cells
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PC12 cells (#CRL-1721, ATCC, USA), a rat pheochromocytoma cell line derived from the adrenal medulla (#CRL-1721, ATCC, USA), were seeded on a dish, which was coated by poly l -lysine (0.01%; Sigma Chemical Co., USA), and grown in RPMI 1640 medium (#11875093, Gibco, USA), supplemented with 10% heat-inactivated horse serum (#26050088, Gibco, USA), 5% fetal bovine serum (FBS) (#SH30919.03, Hyclone, USA), and 1% penicillin/streptomycin at 37°C with 5% CO2. For neuronal differentiation, PC12 cells were plated at a density of 1.25x103 cell/cm2 into 96-well plates coated with collagen 1 (#354236, BD Biosciences, USA) and grown in RPMI 1640 medium supplemented with 50 ng/ml nerve growth factor (NGF) (BD Biosciences, USA) and 0.5% FBS for 10 days. The NGF-supplemented media was replaced with fresh media every 3 days during neuronal differentiation.
HT-22 cells (kindly provided by Prof. Gil-Saeng Jeong in Keimyung University), an immortalized mouse hippocampal cell line, were cultured at 37°C and 5% CO2 in Dulbecco's modified eagle medium (DMEM, Invitrogen, USA) supplemented with 10% FBS and 1% penicillin/streptomycin at 37°C and 5% CO2.
HT-22 cells (kindly provided by Prof. Gil-Saeng Jeong in Keimyung University), an immortalized mouse hippocampal cell line, were cultured at 37°C and 5% CO2 in Dulbecco's modified eagle medium (DMEM, Invitrogen, USA) supplemented with 10% FBS and 1% penicillin/streptomycin at 37°C and 5% CO2.
9
Glycolytic Flux Measurement in Cell Types
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Glycolysis in the same number of live cells was measured using the XF Glycolysis Stress Test kit according to the manufacturer's instructions (Agilent). The glycolysis kit directly measures extracellular acidification rate (ECAR) and evaluates the glycolytic flux. In brief, cells originating from the muscle or liver were seeded on wells of XF96 microplates coated with collagen1 (BD Biosciences). Cells originating from the lungs were seeded on wells coated with poly-d -lysine (Sigma-Aldrich), and cells originating from the lymph nodes were plated on wells coated with gelatin (Sigma-Aldrich). Cells were plated at 3 × 104 cells/well 24 to 48 hours prior to the assay. Total RNA was extracted using the RealTime Ready Cell Lysis Buffer (Roche) and subjected to qPCR. ECAR values were normalized to Gapdh mRNA in each sample.
10
Mitochondrial Respiration Profiling
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The oxidative phosphorylation kit measures key parameters of mitochondrial function by directly measuring the oxygen consumption rate (OCR). In brief, the same number of cells originating from the muscle or liver was seeded on collagen 1 (BD Biosciences)–coated wells of XF96 microplates. The same number of cells originating from the lung was seeded on poly-d -lysine (Sigma)–coated wells, and cells originating from the lymph were plated on gelatin (Sigma)-coated wells. Cells were plated at 30,000 cells/well 24 to 48 hours prior to the assay. Total RNA was extracted using “RealTime Ready Cell Lysis Buffer” (ROCHE) and subjected to qPCR. ECAR and OCR values were normalized to Gapdh mRNA for each sample. Each data point represents mean ±SD (n > 4).
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