Blood samples (30 mL) were obtained from nine patients at baseline (Pre), and at the third and sixth month during therapy (Post 3 and Post 6). For one single patient, samples were collected only at baseline and at 3‐month therapy. Blood was processed within 1 h from withdrawal. PBMCs were separated by Ficoll gradient (Leuco‐sep tubes, ThermoFisher Scientific) and viable cells stored in liquid nitrogen until use, or frozen in Qiazol (Qiagen) for RNA extraction and gene expression profiling.
Ficoll gradient
Manufactured by Thermo Fisher Scientific
Ficoll gradient is a centrifugation medium used for the separation and purification of cells, organelles, and other biological particles. It creates a density gradient that allows the separation of different components based on their density.
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Lab products found in correlation
5 protocols using ficoll gradient
1
Blood Sampling for PBMC Analysis
2
Blood Sampling for PBMC Analysis
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Blood samples (30 ml) were obtained from 9 patients at baseline (Pre), and at the 3 rd and 6 th month during therapy (Post 3 and Post 6). For one single patient, samples were collected only at baseline and at 3 months therapy. Blood was processed within 1 hour from withdrawal.
PBMCs were separated by Ficoll gradient (Leuco-sep tubes, ThermoFisher Scientific) and viable cells stored in liquid nitrogen until use, or frozen in Qiazole (Qiagen) for RNA extraction and gene expression profiling.
PBMCs were separated by Ficoll gradient (Leuco-sep tubes, ThermoFisher Scientific) and viable cells stored in liquid nitrogen until use, or frozen in Qiazole (Qiagen) for RNA extraction and gene expression profiling.
3
Isolation and Polarization of M2-like Macrophages
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Buffy coat was mixed 1:2 with PBS. The mixture was then added 3:1 to Ficoll gradient (Invitrogen, 17-1440-03) and spun down at 1500 r.p.m. for 25 min at RT (w/o brakes). The leukocyte ring was collected and washed with PBS. Cells were then resuspended in 0.5% bovine serum albumin in PBS containing anti-CD14 microbeads (130-050-021, Milthenyi Biotec) and purified for positive selection using LS MACS column system (130-042-401, Miltenyi Biotec). The purity of CD14+ cells was analyzed by FACS using an anti-CD14-BV650 antibody (eBioscience). The CD14+ cells were polarized to M2-like macrophages in RPMI medium, 10% FBS, 50 ng/ml granulocyte-macrophage colony-stimulating factor (R&D Systems, 215-GMP), 50 ng/ml macrophage colony-stimulating factor (R&D Systems, 216-GMP), 20 ng/ml interleukin-13 (200-13, PeproTech), and 20 ng/ml of IL4 (200-04, PeproTech) for 7 days. In the experiments with CSS, CSS 1 nM DHT, or CSS 1 µM GSK126 fresh medium and LNCaP-CM, the CD14+ cells were polarized to M2-like macrophages using the same cocktail of cytokine.
4
Derivation of Macrophage-Like Cells
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Buffy coat was mixed 1:2 with PBS. The mixture was then added 3:1 to Ficoll gradient (Invitrogen, 17-1440-03) and spun down at 2100 rpm for 25 min at RT (w/o brakes). The leucocyte ring was collected and washed with cold PBS. Cells were then resuspended in MACS buffer (0.5% BSA in PBS) containing anti-human CD14 microbeads (Miltenybiotec). CD14+ cells were cultured in RPMI medium, 10% FBS, 20 ng ml−1 GM-CSF (R&D Systems, 215-GM-010) and 1% Pen Strep (Gibco) (FBS-RPMI) for 24 h. Then, medium was replaced with RPMI, 5% DCC, 20 ng ml−1 GM-CSF and 1% Pen Strep for 3 days. Cells were then stimulated for 24 h with 10 ng ml−1 of IFN-γ (R&D Systems, 285-IF-100) and 10 ng ml−1 of LPS (ENZO, Life Science O55:B5) for differentiation into macrophage-like cells.
5
Isolation and Culture of Ayu Monocytes/Macrophages
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Ayu specimens were deeply anaesthetized with ethylene glycol monophenyl ether (0.03% v/v) before the head kidneys were collected aseptically. MO/MΦ from head kidneys were isolated as previously described [26 (link)]. Briefly, single cell suspensions of head kidneys were prepared using a 100-μm wire mesh, layered using a Ficoll gradient (Invitrogen, Shanghai, China), and subsequently centrifuged at 400 × g for 25 min. The leukocyte fraction in the Ficoll-medium interface was collected and incubated overnight at 24°C. Non-adherent cells were washed off, and the attached cells were incubated with RPMI 1640 medium containing 5% fetal bovine serum (FBS), 5% ayu serum, 100 U/ml penicillin, and 100 μg/ml streptomycin throughout the experiment. Over 95% of the adherent cells were MO/MΦ according to morphological characteristics observed after Giemsa staining. For cAMP blockade, 30 μM Rp-adenosine 3’,5’-cyclic monophosphorothioate triethylamine salt (Rp-cAMPS) was added to culture medium 2 h before stimulation with PGE2.
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