Cells (1.5 × 105/well) were seeded in 24-well plates overnight in serum-containing culture media and then, the media was replaced by serum-free media. Cells were treated with vehicle (ethanol) or indicated concentrations of RCE and the conditioned media was collected at 24 h. The level of VEGF therein was measured using a VEGF enzyme-linked immunosorbent assay kit (R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s protocol. The optical density at 570 nm of each sample was measured using an AMP Platos R 496 microplate reader (AMP Diagnostics, Poland). The proteins present in the conditioned media were concentrated using the Amicon Ultra-0.5 protein purification and concentration column (Millipore) and protein concentration was assayed using the BCA protein assay kit (Thermo Scientific). Levels of VEGF were normalized to the total protein level in each sample. The assays were performed in triplicates and three independent experiments were performed. Data are presented as mean values ± SEM.
Amicon ultra 0.5 protein purification and concentration column
Manufactured by Merck Group
The Amicon Ultra-0.5 is a protein purification and concentration column. It is designed to facilitate the separation and concentration of proteins from small sample volumes. The column utilizes a centrifugal filtration process to isolate and concentrate target proteins.
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4 protocols using amicon ultra 0.5 protein purification and concentration column
1
VEGF Secretion and Protein Quantification
2
Quantifying Secreted MMP-9 Levels
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Cells were seeded in 6-well plates in the presence of vehicle (DMSO) or carnosol for 24 h. The conditioned medium was collected and the levels of secreted MMP-9 were determined using immunoassay kit (Abcam, Cambridge, UK) according to the manufacturer's protocol. The proteins present in the conditioned media were concentrated using the Amicon Ultra-0.5 protein purification and concentration column (Millipore). Levels of MMP-9 were normalized to the total protein level in each sample. The assays were performed three times in triplicates. Data are presented as mean values ± SEM.
3
Quantifying MMP-2, MMP-9, and PgE2 secretion in MDA-MB-231 cells
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MDA-MB-231 cells (2× 105/well) grown to a sub-confluent level in 6-well plate, then cultured for 24 hours in the presence or absence of OSE. The conditioned medium from OSE (0.8 and 1.2 mg/mL) treated cells and untreated cells was collected and the levels of secreted MMP-2, MMP-9 (Invitrogen, Camarillo, CA, USA) or PgE2 (Cayman Chemical, MI, USA) were determined using colorimetric ELISA kitsas per the manufacturer’s instruction. The optical density of each sample was measured using an AMP Platos R 496 microplate reader (AMP Diagnostics, Poland). The proteins present in the conditioned media were concentrated using the Amicon Ultra-0.5 protein purification and concentration column (Millipore) and protein concentration was assayed using the BCA protein assay kit (Thermo Scientific). Levels of the PgE2 and MMPs were normalized to the total protein level in each sample.
4
Quantification of Inflammatory Cytokines
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Cells (2 × 105/well) were cultured in a 6-well plate overnight and then serum-starved for 24 h in the presence of vehicle (ethanol) or indicated concentrations of RCE. Levels of IL-6, −8, and −11 in the collected media were measured using ELISA quantification kit (R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s instructions. For PgE2, culture supernatants were assayed using an ELISA kit from Cayman Chemical (MI, USA). The optical density of each sample was measured using an AMP Platos R 496 microplate reader (AMP Diagnostics, Poland). The proteins present in the conditioned media were concentrated using the Amicon Ultra-0.5 protein purification and concentration column (Millipore) and protein concentration was assayed using the BCA protein assay kit (Thermo Scientific). Levels of the cytokines/PgE2 were normalized to the total protein level in each sample. Assays were performed in triplicates and three independent experiments were performed. Data are presented as mean values ± SEM.
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