Thick ascending limbs from 6- to 8-weeks old male C57BL/6J-HpnHlb320/Hlb320 or C57BL/6J-HpnWT/WT mice were isolated and cultured as reported previously19 (link). Thick ascending limbs were manually collected under a light microscope and seeded on fibronectin-coated (Corning Inc., Corning, NY) 96-well plastic plates (Thermo Fischer Scientific) containing a culture medium based on DMEM:F1219 (link) (Thermo Fischer Scientific) and placed in a humidified chamber at 37 °C and 5% CO2. When cellular outgrow reached near confluence, cells were passed using 0.05% Trypsin-EDTA (Thermo Fischer Scientific) to fibronectin and collagen-coated 0.33 cm2 PTFE filter membranes (Corning Inc., Corning, NY). After a confluent monolayer of cells was reached, supplemented FBS (Thermo Fischer Scientific) was reduced from 2% to 0.1% (v/v) to allow maximal differentiation. Cells were treated with 100 µM bumetanide (Sigma-Aldrich) to the apical side, with 0.1 µM ddAVP (Sigma-Aldrich) to the basolateral side or with 2.5 µg/mL tunicamycin (Sigma-Aldrich) to the apical and basolateral side. Transepithelial voltage measurements were performed using chopstick electrodes (STX2, World precision instruments, Sarasota, FL) according to manufacturer’s instructions.
Fibronectin coated
Manufactured by Corning
Fibronectin-coated lab equipment is a specialized substrate designed for cell culture applications. Fibronectin is a naturally-occurring extracellular matrix protein that promotes cell attachment and growth. The equipment is coated with this protein to provide a favorable environment for culturing various cell types.
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Lab products found in correlation
2 protocols using fibronectin coated
1
Thick Ascending Limb Cell Culture
2
Wound Closure Dynamics in Primary Keratinocytes
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Primary keratinocytes were plated on fibronectin-coated (10 µg/ml; Corning) glass-bottomed six-well culture plates (MatTek). The cells were allowed to adhere to the plates overnight, and the resulting monolayer was then scratched with a pipette tip. Wound closure was monitored every 20 min for 16 h with an inverted Andor-driven spinning-disk confocal system equipped with a Zyla 4.2 (Andor Technology) camera in brightfield conditions with a 10× 0.3 NA air objective. During imaging, cells were maintained at 37°C under an atmosphere containing 5% CO2. The images were subjected to automated analysis with the MiToBo plugin in ImageJ (National Institutes of Health) to determine the cell-free area, which was normalized relative to the initial cell-free area and expressed as a percentage. The start and endpoint images were also manually analyzed by measuring the total cell-free area with the freehand tool. The data shown are the means for two to three experiments with at least six data points per experiment.
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