Thirteen BT cell lines (BT-1 and BT-A–L) were established from in vitro matured and in vitro fertilized blastocysts as described previously [18 (link),27 (link)]. The BT cell lines were cultured and maintained according to a previously described method [28 (link)]. In brief, the cells were cultured in Dulbecco’s modified Eagle’s medium/F-12 medium (Sigma, Saint Louis, MI, USA) containing 100 IU/ml of penicillin and 100 μg/ml of streptomycin (Sigma), supplemented with 10% fetal bovine serum (FBS; Biowest, Nuaillé, France), at 37°C in an atmosphere of 5% CO2. The medium was changed every 2–3 d. A monolayer of confluent BT cells was mechanically passaged by pipetting. Collagen-coated flasks were prepared by incubating flasks with acid-soluble porcine type I collagen (3 mg/ml of type I-C collagen; Nitta Gelatin Osaka, Japan), diluted 10-fold with distilled water and poured into flasks for more than an hour. Flasks were then washed with general culture medium. After a phosphate-buffered saline (PBS) wash, dissociated cell aggregates were plated in the collagen-coated flasks. The cell cultures grown in collagen-coated flasks were used for RNA purification, Western blotting, or immunocytochemistry. BT-1 was used as a standard cell line because it was maintained and monitored for more than 300 passages, and may show features of stable trophoblastic cells.
Collagen type 1 c
Manufactured by Nitta Gelatin
Sourced in Japan, United States
Collagen type I-C is a laboratory product derived from natural sources. It serves as a key component in various research and testing applications. The product provides the core function of a purified form of collagen type I, a common structural protein found in connective tissues.
Automatically generated - may contain errors
Lab products found in correlation
Polycarbonate filter with 8 μm pores, by Neuro Probe (2 mentions)
Pet membrane 24 well 8.0 μm pore size cell culture inserts, by BD (2 mentions)
F12 medium, by Merck Group (1 mentions)
Penicillin, by Merck Group (1 mentions)
Streptomycin, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Biowest (1 mentions)
Cell culture insert, by BD (1 mentions)
Bovine serum albumin bsa, by Fujifilm (1 mentions)
Polybrene, by Nacalai Tesque (1 mentions)
35 mm glass base dishes, by Iwaki (1 mentions)
Penicillin, by Nacalai Tesque (1 mentions)
Streptomycin, by Nacalai Tesque (1 mentions)
Lenti x 293t cells, by Takara Bio (1 mentions)
Dulbecco modified eagle medium (dmem), by Nacalai Tesque (1 mentions)
Fetal bovine serum (fbs), by Merck Group (1 mentions)
Puromycin, by InvivoGen (1 mentions)
48 well micro chemotaxis boyden chamber, by Neuro Probe (1 mentions)
Heparin sodium, by Fujifilm (1 mentions)
Pepsin from porcine gastric mucosa, by Merck Group (1 mentions)
2 aminoethanol, by Fujifilm (1 mentions)
13 protocols using collagen type 1 c
1
Establishment and Maintenance of BT Cell Lines
2
Cell Invasion Quantification Protocol
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The cells were seeded in triplicate onto cell culture inserts (8.0 µm pore size; BD Falcon, Franklin Lakes, NJ, USA) coated with type I‐C collagen (Nitta Gelatin, Osaka, Japan). Twenty‐four h later, we removed cells that had not invaded into the lower surfaces of the filters using cotton swabs and fixed cells that had invaded into the lower surfaces with acetone and methanol (1 : 1), followed by staining with Trypan Blue. Invasion was quantified by visually counting the photographed cells in several fields, followed by statistical analysis.
3
Transwell Assay for Cell Migration
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Cell migration was assessed using a 48-microchemotaxis chamber (Neuro Probe, Maryland, USA) and a polycarbonate filter with 8-μm pores (Neuro Probe). The lower side of the filter was precoated with 10 μg/mL collagen type I-C (Nitta Gelatin, Osaka, Japan). A medium containing 10% FBS was placed in the bottom chamber. Non-irradiated and irradiated cells were trypsinized, washed twice with serum-free medium and suspended in serum-free medium supplemented with 0.1% bovine serum albumin (BSA; FUJIFILM Wako Pure Chemical Corporation). Fifty microliters of cell suspension containing 1.1 × 105 MDA-MB-231 cells were added to the upper wells, and incubated for 3 hours. The cells were fixed with 4% paraformaldehyde and stained with hematoxylin. The number of cells that migrated to the lower side through the pores was counted at ×40 magnification in three independent fields. At least three experiments were conducted.
4
Lentiviral Production and Cell Line Generation
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HeLa cells were obtained from the Cell Resource Center for Biomedical Research, Institute of Development, Aging and Cancer, Tohoku University. Lenti-X-293T cells were purchased from Clontech Laboratories. All cells were maintained in Dulbecco's modified Eagle's medium (DMEM) (Nacalai Tesque) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Sigma), 100 U/mL penicillin, and 100 μg/mL streptomycin (Nacalai Tesque) at 37°C under a humidified 5% CO2 atmosphere. For live cell imaging, cells were seeded on 35 mm glass-base dishes (Iwaki) coated with collagen type I-C (Nitta Gelatin).
For lentiviral production, Lenti-X-293T cells were cotransfected with the pCSIIpuro-mNG-eDHFR(69K6), psPAX2 (Addgene plasmid #12260, a gift from D. Trono), and pCMV-VSV-G-RSV-Rev (RIKEN BioResource Research Center plasmid RDB04393, provided by Dr. Miyoshi, Keio University) plasmids 45 using a polyethlenimine "Max" (Polysciences Inc.). Virus-containing media were collected 48 h after transfection, filtered, and used to infect target HeLa cells with 8 μg/mL polybrene (Nacalai Tesque). Infected HeLa cells were selected with 0.5 μg/mL puromycin (InvivoGen) for at least 7 days. Bulk populations of selected cells were used.
For lentiviral production, Lenti-X-293T cells were cotransfected with the pCSIIpuro-mNG-eDHFR(69K6), psPAX2 (Addgene plasmid #12260, a gift from D. Trono), and pCMV-VSV-G-RSV-Rev (RIKEN BioResource Research Center plasmid RDB04393, provided by Dr. Miyoshi, Keio University) plasmids 45 using a polyethlenimine "Max" (Polysciences Inc.). Virus-containing media were collected 48 h after transfection, filtered, and used to infect target HeLa cells with 8 μg/mL polybrene (Nacalai Tesque). Infected HeLa cells were selected with 0.5 μg/mL puromycin (InvivoGen) for at least 7 days. Bulk populations of selected cells were used.
5
Cell Adhesion Assay Protocol
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Cell adhesion assays were performed as described previously (Hieda et al., 2015 (link)). Briefly, 96-well plates were coated with 100 μL of 5 μg/ml laminin (AGC Inc., Tokyo, Japan), 5 μg/ml vitronectin (Wako Pure Chemical, Osaka, Japan), 5 μg/ml fibronectin (AGC Inc.), 5 μg/ml collagen type IC (Nitta Gelatin, Osaka, Japan), or 3% bovine serum albumin (BSA) and blocked with 3% BSA. Next, cells were added to the plates. After 2 h of incubation at 37°C, plates were washed with phosphate-buffered saline (PBS) and cells were stained with crystal violet. The absorbance was measured using measurement filter 595 nm and reference filter 630 nm. Experiments were repeated at least four times.
6
Cell Migration Assay Protocol
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Cell migration assays were performed as previously described using a 48‐well microchemotaxis Boyden chamber (Neuro Probe, Gaithersburg, MD, USA) and polycarbonate filter with 8‐μm pores (Neuro Probe).30 The lower side of the filter was precoated with 10 μg/mL collagen type I‐C (Nitta Gelatin, Osaka, Japan). After incubation for 3 hours, the number of cells that had migrated to the lower side was counted in 3 independent fields. These experiments were repeated a minimum of 4 times.
7
Boyden Chamber Cell Invasion Assay
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Boyden chamber migration assays were conducted using transparent PET membrane 24‐well 8.0‐μm pore size cell culture inserts (BD Falcon, Franklin Lakes, NJ, USA) coated with collagen type I‐C (Nitta Gelatin, Osaka, Japan). After the cells were seeded in triplicate on the inserts, the cells that had not invaded the lower surfaces of the filters were removed from the upper faces of the filters using cotton swabs. Cells that had invaded into the lower surfaces of the filters were fixed in methanol and acetone and stained with Giemsa. Invasion was quantitated by visually counting the photographed cells, and evaluated by statistical analysis.
8
Healthy Porcine Liver Extraction
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The materials and reagents in this study were obtained from the following sources: heparin sodium (1,000,000 units), albumin from bovine serum (BSA, Cohn Fraction V, pH 7.0), and 2-Aminoethanol were purchased from Wako Pure Chemicals (Osaka, Japan). Gelatin from porcine skin (gel strength 300 Type A), Triton X-100, pepsin from porcine gastric mucosa (lyophilized powder, ≥2500 units/mg protein), and Alcian blue 8GX were purchased from Sigma Aldrich Ltd (St. Louis, MO, USA). Collagen type I-C was acquired from Nitta Gelatin Inc. (Osaka, Japan) and collagen quantitation assay kit was acquired from Cosmo Bio (Tokyo, Japan). All other chemicals were of analytical grade. The healthy porcine liver was harvested from adult pigs, Fukuokashokunikuhanbai Co. Ltd. (Fukuoka, Japan).
9
Hepatic Differentiation of DFAT Cells
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DFAT cells were cultured under a humidified atmosphere of 5% CO2 and 95% air at 37°C on collagen type I‐C (Nitta Gelatin)‐coated tissue 40‐mm culture dishes (TPP) containing Dulbecco's modified Eagle's medium (DMEM) (Nissui Pharmaceutical) supplemented with 10% fetal bovine serum (FBS) (Moregate BioTech). DFAT cells were grown to semiconfluence for differentiation. Hepatic differentiation was induced by changing the medium to SHM + YAC (Chen et al., 2012 ), namely DMEM/F12 (Gibco) containing 2.4 g/L NaHCO3 and l ‐glutamine, which was supplemented with 5 mM HEPES (Sigma‐Aldrich), 30 mg/L l ‐proline (Sigma‐Aldrich), 0.05% BSA (Sigma‐Aldrich), 10 ng/ml epidermal growth factor (PeproTech), insulin–transferrin–serine (ITS)‐X (Gibco), 10–7 M Dexamethasone (Dex)(Sigma‐Aldrich), 10 mM nicotinamide (Wako), 1 mM ascorbic acid‐2 phosphate (Sigma‐Aldrich), 10 mM Y‐27632 (Wako), 0.5 mMA83‐01 (Wako) and 3 mM CHIR99021 (Axon Medchem). After 2 days, the culture medium was replaced with SHM + YAC, which was supplemented with 20 ng/ml oncostatin M (OsM) (Wako), 10−6 M Dex and 500 ng/ml R‐spondin1 (PeproTech). The cells were then cultured for 12 days, with fresh medium provided every 2 days.
10
Primary Hepatocyte Isolation from Mouse Liver
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Primary hepatocytes were isolated from adult mouse livers by standard two‐step collagenase digestion. Briefly, the liver was preperfused through the portal vein with calcium‐ and magnesium‐free Hank's/EGTA solution and then perfused with approximately 40 ml of Hank's solution containing 0.05% collagenase (Sigma‐Aldrich). All perfusion solutions were preheated at 37°C. The liver was removed and was later placed in a dish with Hank's solution and agitated gently after opening the liver capsule. The suspension of released cells was filtered through a 100‐µm nylon mesh to remove cell clumps. The cell suspension was then transferred to 50‐ml tubes, which were filled with Hank's solution, and the cells were collected via centrifugation at 50 g for 7 min. Then, the cells were resuspended in Percoll buffer (90% Percoll (GE Healthcare), 1 × Hank's), and dead cells were removed via centrifugation at 50 g for 15 min. The cells were washed in 40 ml/tube of Hank's solution twice via centrifugation at 50 g for 2 min. The purified hepatocytes were used for various experiments. Isolated cells were seeded at 2.2 × 104 cells/cm2 in SHM containing 10% FBS onto collagen type I‐C (Nitta Gelatin)‐coated 40‐mm tissue culture dishes (TPP).
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