A scratch assay was conducted to determine the migratory ability of hPASMCs in each group. Briefly, pretreated cells were incubated with serum-free medium with mitomycin C (1 μg/ml, M0503, SIGMA) for 1 h. Subsequently, a scratch was made in the cells using a pipette tip. Afterward, the cells were exposed to hypoxia (2% O2, 93% N2, and 5% CO2) or normoxia for 24 h. Representative images were captured at time points of 0 h and 24 h to measure cell migration.
M0503
Manufactured by Merck Group
The M0503 is a laboratory equipment product offered by the Merck Group. It is designed for general laboratory use. The core function of the M0503 is to assist in the handling and processing of samples and other materials in a controlled laboratory environment.
Automatically generated - may contain errors
Lab products found in correlation
Facscalibur, by BD (1 mentions)
P4864, by Merck Group (1 mentions)
M0255, by Duchefa Biochemie (1 mentions)
Woundmaker, by Sartorius (1 mentions)
Incucyte s3 system, by Sartorius (1 mentions)
Wound maker, by Merck Group (1 mentions)
α mem, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Non essential amino acids (neaa), by Thermo Fisher Scientific (1 mentions)
Glutamine, by Merck Group (1 mentions)
Transferrin, by Merck Group (1 mentions)
Mitomycin, by Merck Group (1 mentions)
P8783, by Merck Group (1 mentions)
I1882, by Merck Group (1 mentions)
Putrescine, by Merck Group (1 mentions)
M3148, by Merck Group (1 mentions)
F0291, by Merck Group (1 mentions)
Eclipse te300, by Nikon (1 mentions)
Etoposide, by Merck Group (1 mentions)
Phleomycin, by InvivoGen (1 mentions)
7 protocols using m0503
1
Assessing hPASMC Migration under Hypoxia
2
Assessing BRCA Pathway Integrity
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To challenge the FA/BRCA pathway, LCLs were submitted to increased concentrations of MMC (0–1000 nM, M0503, Sigma-Aldrich) in fresh medium for a period of 120 h. After this period, cells were resuspended in phophate-buffered saline (PBS)–bovine serum albumin (BSA; 0.05%) containing 0.5 mg/mL propidium iodide (P-4864, Sigma-Aldrich) and incubated for 10 min at 4 °C. Cell viability was determined by flow cytometry based on the PI exclusion test. The analysis was carried out, taking into account the viability of the 0–3 nM cells as a reference in each cell type condition. All the doublets were discarded from the analysis. Flow cytometry analysis was performed on an FACS Calibur (Becton-Dickinson, San Jose, USA). As internal controls, a healthy donor (normal control) was used in each assay, along with three FA patients (positive controls in one of the experiments (kindly provided by Dr Juan Bueren´s laboratory).
3
Seedling Surface Sterilization and Treatment
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The seeds were surface-sterilized with 0.05% SDS, 70% ethanol for 5 min, rinsed 5 min with 95% ethanol, air dried and evenly spread on sterile 0.8% agar, 1% sucrose and 1X Murashige and Skoog (M0255; Duchefa Biochemie) medium supplemented with or without the indicated concentrations of MMC (M0503; Sigma-Aldrich) or CP (TEVA Pharma). Whole seedlings were harvest at 14-day-old for analysis.
4
Microglia and MBM Cells Migration Assay
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Microglia cells were seeded on a collagen-coated 96-well plate. On confluence, the cell monolayer was scratched using a 96-well WoundMaker (Essen BioScience, Inc.). Cells were washed twice and growth medium was applied on the cells (a mixture of RPMI 1640 and Prigrow III culture media (1:1) supplemented with 10% FCS) for 48 h.
MBM cells were seeded on a collagen-coated 96-well plate. On confluence, the cells were treated with mitomycin C (10 µg/mL; M0503, Sigma-Aldrich) for 3 h, after which the cell monolayer was scratched using a 96-well WoundMaker. Cells were washed twice and microglia-conditioned medium (CM) or starvation medium was applied on the cells for 48 h.
The wounds were imaged every 3 h for 48 h, and images were analyzed using the IncuCyte S3 system (Essen BioScience, Inc.). Each experiment was repeated at least three times.
MBM cells were seeded on a collagen-coated 96-well plate. On confluence, the cells were treated with mitomycin C (10 µg/mL; M0503, Sigma-Aldrich) for 3 h, after which the cell monolayer was scratched using a 96-well WoundMaker. Cells were washed twice and microglia-conditioned medium (CM) or starvation medium was applied on the cells for 48 h.
The wounds were imaged every 3 h for 48 h, and images were analyzed using the IncuCyte S3 system (Essen BioScience, Inc.). Each experiment was repeated at least three times.
5
Murine CD1 SSC Cell Culture Protocol
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The CD1 SSC cell line from mice was donated by professor Wu Ji’s laboratory from Shanghai Jiao Tong University. The culture medium was based on Minimum Essential Medium α (MEM-α) (12571-063, Gibco, Grand Island, NY, USA) containing 2 mM glutamine (G7012, Sigma, MO, USA), 10% foetal bovine serum (FBS) (16000-36, Gibco), 0.5× pen/strep (15240-062, Invitrogen, Grand Island, NY, USA), 1× nonessential amino acid (NEAA) (11140-050, Gibco) solution, 1× β-mercaptoethanol (β-ME) (M3148, Sigma), 25 μg/ml insulin (I1882, Sigma), 100 μg/ml transferrin (T1428, Sigma), 60 μM putrescine (P5780, Sigma), 60 ng/ml progesterone (P8783, Sigma), 40 ng/ml glial cell line-derived neurotrophic factor (GDNF) (512-GF-050, R&D Systems, Minneapolis, MN, USA) and 3–5 ng/ml basic fibroblast growth factor (bFGF) (F0291, Sigma). The feeder layer cells were STO cells treated with mitomycin (M0503, Sigma). The SSCs were incubated at 37 °C in the presence of 5% CO2.
6
Mitomycin C-Inhibited Wound Healing Assay
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Confluent cells were pre-treated with mitomycin C (25 μg/ml, Sigma-Aldrich M0503) for 30 min to inhibit cell growth. Then, a straight scratch was performed and the medium replaced by a fresh one without serum (for MEFs) or with 2% FBS (for HCT116 cells). Cells were maintained for 8h-48h at 37°C and 5% CO2. Migration was followed by a phase-contrast microscope (Eclipse TE300 Nikon coupled to a digital camera) at different time points. Photographs were taken to quantify (using TScratch program) the percentage of wound healing closure at the different times.
7
Cytotoxicity Screening of DNA-Damaging Agents
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Cells were plated at 25,000 per well in 48 wells plates and imaged for 7 days using the Incucyte Live-Cell Analysis System upon exposure to radiation (X-ray) or increased doses of phleomycin (invivoGen, 11006-33-0), MMC (Sigma, M0503) or etoposide (Sigma, E1383).
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