Human aortic smooth muscle cells (HAoSMC, PromoCell, Heidelberg, Germany) were grown in culture flaks using SMC medium 2 (PromoCell) to 80% confluency at 37°C in humidified air containing 5% CO2. Passaging was performed with the detachment kit (PromoCell) at a ratio of 1:4. All HAoSMCs were studied between the fifth and ninth passage. 5x104 cells per well in 1 mL medium were grown for 24 h before further tests. A cell proliferation kit (CFSE, PromoCell) was used to measure proliferation of HAoSMCs. Warmed sterile Dulbecco´s PBS (PAA Cell Culture Company, Cambridge, USA) mixed with 5 μM CFSE was added to 5x104 HAoSMC/mL. Cell suspensions were added on cover slip glass in 24-well plates with or without a material disc positioned in the center of the wells. Cells on the surface of the material discs (surface contact) and cells periphery of the 24-well plates not in contact with the discs (no contact) were fixed with 4% paraformaldehyde (PFA) in PBS. HAoSMC apoptosis was determined using propidium iodide solution (BD Biosciences, USA) with staurosporine (PromoCell) used as positive control. HAoSMC nuclei were stained with DAPI mounting medium (Vectashield, Vector, USA). Proliferating cells in three identically-sized areas of the center (disc surface) and in three areas of the periphery of the 24-well plates (no contact) were counted using fluorescence microscopy (AxioVision).
Staurosporine
Manufactured by PromoCell
Sourced in United States
Staurosporine is a laboratory reagent used as a protein kinase inhibitor. It is a potent and broad-spectrum inhibitor of various protein kinases, making it a valuable tool for researchers investigating cell signaling pathways.
Automatically generated - may contain errors
2 protocols using staurosporine
1
Aortic Smooth Muscle Cell Proliferation
2
Colonic Apoptosis and Permeability Assessment
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Colons from C57BL/6 NRJ mice were removed and washed 3 times in cold Krebs’s solution. For immunostaining, colon pieces were incubated with biopsy medium (Dulbecco modified Eagle medium containing 4.5 g/L glucose, 2.5% fetal bovine serum, 100 IU/mL penicillin, 100 μg/mL streptomycin, 20 μg/mL gentamycin, and 1.1 μg/mL AmphoB) containing 10 μmol/L iloprost at 37°C with 95% O2 and 5% CO2. After 1 hour, 1 μmol/L staurosporine (Promocell, Heidelberg, Germany) was added to colon segments for 2 hours before fixing tissues for 1 hour in 4% paraformaldehyde in PBS. Apoptosis scores were assessed on these samples by quantifying cells positive for cleaved caspase-3 staining. The extent of apoptosis was scored as 0–2 (0 = no positive cells, 1 = 1/2 positive basal cells, 2 = positivity along the entire crypt). An extended score factor of 1–4 was applied when positivity reached 25%, 50%, 75%, or 100% of the fragment analyzed, respectively. For the paracellular permeability measurement, 8 pieces of each colon were incubated with biopsy medium containing 1–100 μmol/L iloprost (Figure 7 ) as described earlier. After 4 hours, 1 μmol/L staurosporine was added to colon segments and incubated overnight. Twenty-four hours after the start of the incubation process, colon pieces were mounted in Ussing chambers, and paracellular permeability was measured as detailed above.
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