Cells were plated on glass coverslips coated with 50 µg/ml human type IV collagen (C7521; Sigma-Aldrich) 24 h before imaging, at 15% density (∼200,000 cells) for HGF stimulation of subconfluent colonies, or at confluence (∼2 million cells) for wound healing assays and HGF experiments on confluent monolayers. Cells were exposed to HGF 12 h after starvation and imaged at t = 0, and then every 20 min for 4–7 h (usually 5 h) after HGF addition. Wound healing assays were performed either scratching the monolayer with a 200-µl pipette tip and washing detached cells or removing a two-well silicone insert (Ibidi). For isolated cell experiments, cells were plated 8 h before imaging and starved for 2–3 h before adding HGF, to minimize cell doublets caused by cell division. Live cells were imaged in FluoroBrite DMEM medium (Life Technologies) without phenol red supplemented with 10% or 0.5% FBS depending on experiments, 1 U/ml penicillin, 20 mM Hepes, and 2.5 mM l -glutamine, at 37°C, and 5% CO2.
C7521
Manufactured by Merck Group
Sourced in United States
The C7521 is a laboratory equipment product. It is designed for use in research and scientific applications. The core function of the C7521 is to perform tasks related to the analysis and processing of samples.
Automatically generated - may contain errors
Lab products found in correlation
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Two well silicone insert, by Ibidi (1 mentions)
Fluorobrite dmem medium, by Thermo Fisher Scientific (1 mentions)
P4170, by Merck Group (1 mentions)
C7774, by Merck Group (1 mentions)
A3782, by Merck Group (1 mentions)
H7379, by Merck Group (1 mentions)
Dmem ham s f12, by Thermo Fisher Scientific (1 mentions)
Pyruvate, by Thermo Fisher Scientific (1 mentions)
Egf af 100 15, by Thermo Fisher Scientific (1 mentions)
Rpmi 1640 glutamax medium, by Thermo Fisher Scientific (1 mentions)
Fgf 100 18b, by Thermo Fisher Scientific (1 mentions)
2 mercaptoethanol, by Thermo Fisher Scientific (1 mentions)
Hepes, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Axiovert 200, by Zeiss (1 mentions)
Costar 3470, by Corning (1 mentions)
P38 mapk 9212, by Cell Signaling Technology (1 mentions)
Amersham enhanced chemiluminescence rpn2232, by GE Healthcare (1 mentions)
Anti rabbit igg hrp linked antibody, by Cell Signaling Technology (1 mentions)
9 protocols using c7521
1
Collagen-Mediated HGF Stimulation of Cells
2
Quantifying Adherent Podocytes in Culture
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Podocytes were suspended in 2 mL of serum-free culture medium and counted using a hemacytometer. Podocytes (1 × 106) were cultured in six-well plates coated with type IV collagen (C7521; Sigma-Aldrich Corp., St. Louis, MO, USA) and incubated at 37 °C for 2 h. Five wells were used for each experimental group. The plates were washed twice with phosphate-buffered saline (PBS), then adherent podocytes were fixed in 4% paraformaldehyde (PFA), stained with crystal violet, and assessed by microscopy. The numbers of adherent podocytes in each field of view were counted using ImageJ software then the count was averaged by the number of podocytes in five fields of view.
3
Live/Dead Cell Spheroid Staining
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The live/dead staining assay was conducted using a 2 mg/ml propidium iodide stock (Sigma-Aldrich Co. LLC, P4170) at a 1:50 dilution and a 5 mg/ml fluorescein diacetate stock (Sigma-Aldrich Co. LLC, C-7521) at a dilution of 1:625 in PBS. As a control or the dead stain, spheroids were fixed using 4% PFA and stained with the live/dead stain.
4
Evaluation of Protein Interactions
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rSmPOP (0.7 μg) was incubated at 37°C for 16 h with 100 μg of human hemoglobin, human serum albumin, human collagens type I and IV (Sigma-Aldrich, catalog numbers H7379, A3782, C7774 and C7521 respectively) in 100 mM Tris-HCl, pH 8.0, in a final volume of 50 μL. After incubation, a 10 μL sample was resolved by 15% SDS-PAGE or Tricine-SDS-PAGE and stained with Coomassie Brilliant Blue G250.
5
Culture and Maintenance of Cell Lines
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INS-1E cells were obtained from Pierre Maechler and maintained in RPMI 1640 Medium GlutaMAX™ (61870044, Life Technologies—Carlsbad, CA, USA) supplemented with 5% FBS (10500064, Life Technologies), 1% Penicillin/Streptomycin (15070063, Life Technologies), 1 mM Pyruvate (11360-039, Life Technologies), 10 mM HEPES (15630-056, Life Technologies), and 50 µM 2-Mercaptoethanol (31350-010, Life Technologies). NT-3 cells were cultivated in RPMI 1640 Medium GlutaMAX™ with 10% FBS, 1% Penicillin/Streptomycin, 20 ng/mL EGF (AF-100-15, Peprotech – Cranbury, NJ, USA), 10 ng/mL FGF (100-18B, Peprotech) on plates coated with 50 µg/mL H2O Collagen from human placenta (C7521, Sigma-Aldrich—St. Louis, MI, USA), as previously reported [21 (link)]. NT-3 cells carry a homozygous missense mutation of MEN1 (chromosome 11, position 64572018; c.1621A>G; p.T541A) [21 (link)]. The BON1 cells were provided by E.J. Speel, Maastricht, Netherlands and cultured in DMEM/Ham’s F12 (11320033, Life Technologies) with 10% FBS and 1% Penicillin/Streptomycin.
6
Collagen Conjugation and Fluorescent Imaging
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5 mg of type IV human placenta collagen (Sigma-Aldrich, C7521) was reconstituted in 5 mL of phosphate buffer (PBS). The sample was split into 10 0.5 mL aliquots and stored at −20 °C. 1 mg of NHS activated AlexaFluor480 green fluorescent dye (Invitrogen) was dissolved in 200 µL dimethylformamide. Sample was split into 20 10 µL aliquots and stored at −20 °C. 28 µL of AlexaFluor480 (5 mg/mL) was added in a solution of 500 µL of collagen (1 mg/mL). Reactions were allowed to progress on a tube roller for 1 h 15 min at room temperature. Unreacted dye was removed by centrifugal filtration at 3000 RCF, using 10000 MWCO spin filters. Protein was washed three times with water. Dye conjugated collagen was collected from the filter surface after dismantling the filter apparatus by immersion in 1 mL PBS for 5 minutes under agitation (tube roller). Green-labelled protein transferred on HH masks and PAm hydrogels was detected using a fluorescence confocal microscope (Zeiss Axiovert 200).
Fluorescent micrographs of micro patterned FITC-labelled protein were analyzed by using ImageJ software (available athttp://rsbweb.nih.gov/ij/download.html ) to extract the intensity profiles.
Fluorescent micrographs of micro patterned FITC-labelled protein were analyzed by using ImageJ software (available at
7
Isolation and Culture of Human Nasal Epithelial Cells
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Freshly obtained nasal mucosa was dissociated in the 0.1% protease type XIV solution (p5147; Sigma-Aldrich) in DMEM at 4°C. The epithelial cells were released from their tissue matrix by vigorous shaking after overnight incubation. Then the epithelial cells were plated on a plastic culture dish to remove fibroblasts at 37°C in a 5% CO2 atmosphere for 1 h. The purified epithelial cells were seeded on transwell (Corning Costar 3470; Corning Inc.) with human placental collagen (C7521; Sigma-Aldrich). Cells were cultured at 37°C in a 5% CO2 atmosphere until establishing air–liquid interface (ALI) culture by removing the apical culture medium. The DMEM: BEGM (1:1) culture medium containing 50 nM all-trans retinoic acid (R2625; Sigma-Aldrich) was replaced every 2 d. The differentiated HNECs were treated with lentivirus particles and harvested after 2 d for fluorescence microscopy.
8
Western Blot Protein Expression Analysis
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Equal amounts of cell lysates were resolved by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes. After blocking with 1X trishydroxymethylaminomethane‐buffered saline containing 5% weight/volume nonfat dry milk and 0.1% Tween‐20, the membranes were incubated with the primary antibody (overnight at 4°C) followed by horseradish peroxidase (HRP)‐conjugated secondary antibody (1 hour at room temperature). Protein bands were visualized using Amersham enhanced chemiluminescence (RPN2232; GE Healthcare, Piscataway, NJ). Primary antibodies for p44/42 MAPK (9102) and p38 MAPK (9212) were purchased from Cell Signaling Technology (Danvers, MA), and primary antibodies for beta actin (A5316) and type IV collagen (c7521) were purchased from Sigma. Secondary antibodies were anti‐mouse IgG (heavy + light chains) HRP conjugate (81‐6520; Zymed Laboratories, South San Francisco, CA) and anti‐rabbit IgG, HRP‐linked antibody (7047; Cell Signaling Technology).
9
Human Placenta Collagen Reconstitution
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5 mg of type IV human placenta collagen (Sigma-Aldrich, C7521) was reconstituted in 5 mL of sterile 1 X phosphate buffered saline (PBS) and let mix on a roller shaker for 3 hours. The solution was then split into five aliquots of 1 mL and stored at −20 °C.
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