ADSCs (Lonza Group AG, Basel, Switzerland) were co-cultured with normal human dermal fibroblasts (NHDFs) as previously reported28 (link)30 (link). Briefly, NHDFs were seeded in six-well plates (IWAKI, Shizuoka, Japan) and cultured in Dulbecco's modified Eagle's Medium (DMEM) (Gibco, Life Technologies, Carlsbad, CA, USA) containing 10% fetal bovine serum (Sigma-Aldrich, St. Louis, MO, USA) for 24 hours, while ADSCs were seeded on 0.4-µm Millicell® hanging cell culture inserts (Merck Millipore, Darmstadt, Germany) coated with type IV collagen (Nitta Gelatin, Osaka, Japan) and placed onto the plates. All-trans retinoic acid (Sigma-Aldrich) was added at 1 µM to the upper chamber. After culturing for 3 days, 25 ng/ml of bone morphogenetic protein 4 (R&D Systems, Minneapolis, MN, USA) was also added to the upper chamber. After 4 days, the media were replaced with keratinocyte serum-free medium (KSFM) (Thermo Fisher Scientific, Waltham, MA, USA). After 7 days of culture in KSFM, ADSCs were removed from the co-culture system and cultured on a dish coated with type IV collagen in KSFM for an additional 14 days.
Collagen type 4
Manufactured by Nitta Gelatin
Sourced in Japan
Collagen type IV is a type of lab equipment used for the extraction and purification of collagen, a structural protein found in the extracellular matrix of various tissues. It serves as a core component in the assessment and analysis of collagen-related biological processes and characteristics.
Automatically generated - may contain errors
Lab products found in correlation
Fibronectin, by Merck Group (3 mentions)
Laminin, by Merck Group (2 mentions)
Type 1 collagen, by Nitta Gelatin (2 mentions)
Fibronectin, by Fujifilm (2 mentions)
Bone morphogenetic protein 4 (bmp4), by R&D Systems (1 mentions)
Six well plates, by Iwaki (1 mentions)
Adscs, by Lonza (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Merck Group (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Cnt prime medium, by CELLnTEC (1 mentions)
Methylisobutylxanthine, by Merck Group (1 mentions)
Insulin, by Merck Group (1 mentions)
Phorbol 12 myristate 13 acetate pma, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Biowest (1 mentions)
Bemcentinib, by Selleck Chemicals (1 mentions)
Penicillin streptomycin mixture, by Thermo Fisher Scientific (1 mentions)
Chir99021, by Selleck Chemicals (1 mentions)
Growth factor reduced matrigel, by Corning (1 mentions)
Accutase, by Nacalai Tesque (1 mentions)
7 protocols using collagen type 4
1
Co-culture of ADSCs and NHDFs for Keratinocyte Differentiation
2
Immunostaining of Epithelial Cells from Tooth Germs
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Epithelial cells isolated from the mandibular first molar tooth germs of ED14.5 C57BL/6 mice were seeded at a density of 1.5 × 105 cells/cm2 on glass-bottomed dishes coated with 20 µg/mL fibronectin (Sigma) and 100 µg/mL type IV collagen (Nitta Gelatin) and cultured in CnT-Prime medium (CELLnTEC, Bern, Switzerland). On the next day, 500 ng/mL IGF1 was added into the medium. After 4-day culture, cells were fixed using 4% PFA for 15 min and treated with 0.1% Triton-X100 in PBS for 15 min at room temperature. After cells were blocked with 3% bovine serum albumin (Sigma) in PBS for 1 h at room temperature, they were incubated with an anti-AMBN antibody (1:100; Santa Cruz Biotechnology Inc.) overnight at 4 °C. Cells were treated with goat anti-rabbit IgG Alexa 488 (1:1000; Invitrogen) for 1 h at room temperature. Nuclei were stained with DAPI (1:1000; KPL). Signals were visualized using C2si (Nikon Instech). Integrated density measurements of five microscopic fields in each sample were performed using ImageJ software (https://imagej.nih.gov/ij/ ).
3
Cell Differentiation Protocols for PC-12, HL-60, and 3T3-L1
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PC-12 cells (1 × 105 cells) were seeded onto a type-IV collagen (Nitta Gelatin, Osaka, Japan)-coated 35-mm glass bottom dish (Iwaki Glass, Chiba, Japan) with 2 ml of medium and incubated for 24 h prior to differentiation. The cells were then induced to undergo neuronal differentiation in RPMI-1640 medium containing 0.1% horse serum, 0.05% FBS, penicillin/streptomycin, and 50 nM NGF (BD Biosciences).
HL-60 cells (1 × 106 cells) were seeded onto a 100-mm tissue culture dish with 10 ml of medium or a μ-Dish 35-mm, ibiTreat, tissue culture-treated dish (ibidi, Munich, Germany) with 2 ml of medium and incubated for 24 h before differentiation. The cells were then treated with 1 μM all-trans retinoic acid (ATRA) (Sigma-Aldrich, St. Louis, MO) or 16 nM phorbol 12-myristate 13-acetate (PMA) (Sigma-Aldrich) to induce differentiation into neutrophil- or macrophage-like cells for the indicated time.
3T3-L1 cells (1 × 105 cells) were seeded onto a μ-Dish 35-mm, ibiTreat, tissue culture-treated dish and cultured to confluency. After 48 h of culture, the cells were induced to differentiate into adipocytes by incubation in DMEM supplemented with 10% FBS, 1 μM dexamethasone (Wako Chemical Industries, Ltd., Osaka, Japan), 0.5 mM methylisobutylxanthine (Sigma), and 1 μg/ml insulin (Sigma-Aldrich).
HL-60 cells (1 × 106 cells) were seeded onto a 100-mm tissue culture dish with 10 ml of medium or a μ-Dish 35-mm, ibiTreat, tissue culture-treated dish (ibidi, Munich, Germany) with 2 ml of medium and incubated for 24 h before differentiation. The cells were then treated with 1 μM all-trans retinoic acid (ATRA) (Sigma-Aldrich, St. Louis, MO) or 16 nM phorbol 12-myristate 13-acetate (PMA) (Sigma-Aldrich) to induce differentiation into neutrophil- or macrophage-like cells for the indicated time.
3T3-L1 cells (1 × 105 cells) were seeded onto a μ-Dish 35-mm, ibiTreat, tissue culture-treated dish and cultured to confluency. After 48 h of culture, the cells were induced to differentiate into adipocytes by incubation in DMEM supplemented with 10% FBS, 1 μM dexamethasone (Wako Chemical Industries, Ltd., Osaka, Japan), 0.5 mM methylisobutylxanthine (Sigma), and 1 μg/ml insulin (Sigma-Aldrich).
4
Cell Adhesion Assay on ECM Proteins
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Ninety‐six well plates were precoated with 0.3 mg/mL collagen type I (Nitta Gelatin, Osaka, Japan), 0.3 mg/mL collagen type IV (Nitta Gelatin), 50 μg/mL fibronectin (Sigma), FBS (BioWest), or 20 μg/mL laminin (Sigma) and then blocked with DMEM containing 0.5% BSA for 1 h. Thereafter, 2 × 104 cells were seeded onto the coated plates and incubated for 30 min. Unadhered cells were removed by washing with DMEM containing 0.1% BSA. Adherent cells were fixed with 4% paraformaldehyde (PFA), stained with 0.5% crystal violet, and washed. Finally, crystal violet was solubilized with 2% SDS, and absorbance of 595 nm (A595) was determined by a microplate reader.
5
Cell Adhesion Assay on Extracellular Matrix
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For in vitro cell adhesion assay, 96-well plates were pre-coated with either collagen type I (Nitta Gelatin, Osaka, Japan), collagen type IV (Nitta Gelatin), fibronectin (Sigma-Aldrich, St. Louis, MO, USA), laminin (Sigma-Aldrich) or FBS, and then blocked with MEM containing 0.5% BSA. Thereafter, 1 9 10 4 cells were seeded onto the coated plates and incubated for 30 min at 37°C. Unadhered cells were removed, and adherent cells were fixed with paraformaldehyde, stained with crystal violet and washed. Finally, crystal violet was solubilized with sodium dodecyl sulfate, and absorbance at 550 nm was determined using a microplate reader (Dainippon Pharmaceutical).
6
Establishment of an in vitro blood-brain barrier model
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All-trans RA (#0695) and A83-01 (#2939) were obtained from Tocris (Bristol, UK). FGF2 (#AF-100-18B) was obtained from PeproTech (Rocky Hill, NJ, USA). Matrigel Growth Factor Reduced (GFR) (#354230) was obtained from Corning (Corning, NY, USA). Fibronectin (#86088-83-7) was obtained from Fujifilm Wako (Osaka, Japan). Collagen type IV (#638-05921) was obtained from Nitta gelatin (Osaka, Japan). Accutase was obtained from Nacalai Tesque (Kyoto, Japan). ACE2 neutralizing antibody (#AF933, ACE2 inhibitor) was obtained from R&D Systems (Minneapolis, MN, USA). Bemcentinib (#S2841, AXL inhibitor) and CHIR99021 (#S1263) were obtained from Selleck Chemicals (Houston, TX, USA). Meplazumab (#MA5-42304, CD147 inhibitor) and penicillin–streptomycin mixture (#15140122, PS) were obtained from Thermo Fisher Scientific (Waltham, MA, USA). EG00229 (#HY-10799, NRP1 inhibitor) was obtained from MedChemExpress (NJ, USA). Stock concentrations and solvents for chemicals were described in Table 1 . All other reagents were of analytical grade and obtained from commercial sources.
Medium components used in the BBB studies
| Reagent | Stock concentration | Solvent |
|---|---|---|
| All-trans RA | 40 mM | DMSO |
| A83-01 | 10 mM | DMSO |
| FGF2 | 100 µg/mL | water |
| CHIR99021 | 30 mM | DMSO |
| ACE2 Ab | 200 µg/mL | PBS |
| Bemcentinib | 10 mM | DMSO |
| Meplazumab | 930 µg/mL | PBS |
| EG00229 | 100 mM | DMSO |
7
Laminin-based In Vitro Endothelial Barrier
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The E8 fragments of Laminin 211 (LN221F), laminin 411 (LN411F), and laminin 511 (LN511F) were purchased from Nippi, Inc. (Tokyo, Japan). Fibronectin and platelet-poor plasma-derived bovine serum (PDS) were procured from FUJIFILM WAKO (Osaka, Japan). Collagen type IV was purchased from Nitta gelatin Inc. (Osaka, Japan). Human endothelial serum-free medium (HE-SFM), Essential 8 Flex medium, vitronectin (VTN-N), chemically defined lipid concentrate, 1,1′-dioctadecyl-3,3,3′,3′-tetramethyl-indocarbocyanine perchlorate acetylated low-density lipoprotein (Dil-Ac-LDL), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) solution (1 M, pH 7.0–7.6), B-27, Hanks’ balanced salt solution with calcium and magnesium, and lucifer yellow (LY) were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Endothelial Cell Basal Medium 2 was procured from Lonza (Basel, Switzerland). Cell culture inserts (1.0 µm transparent PET membrane) for a 12-well plate and Matrigel GFR were purchased from Corning, Inc. (Corning, NY, USA). Fetal bovine serum (FBS), FITC-dextran 4000 (FD4), and gelatin were procured from Sigma-Aldrich Corporation (St. Louis, MO, USA). TC Protector was purchased from DS Pharma Biomedical (Osaka, Japan). KnockOut Serum Replacement (KSR) was procured from Invitrogen (Carlsbad, CA, USA). A-83-01 was purchased from Cayman Chemical (Ann Arbor, MI, USA)
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