Monocytes were isolated from peripheral blood mononuclear cells (PBMCs) from buffy coats of healthy donors (Sanquin) by a lymphoprep gradient (Axis-Shield) and subsequent percoll gradient centrifugation (Amersham). Informed consent was obtained from all blood donors for the use of their blood. DCs were generated by culturing purified monocytes in RPMI1640 (Invitrogen) supplemented with 10% fetal bovine serum (BioWhittaker), 1000 U/ml penicillin/streptomycin (Lonza), and 2 mM glutamine (Lonza) in combination with IL-4 (262.5 U/ml; Biosource) and GM-CSF (112.5 U/ml; Biosource) for 4–7 days. Ten nanograms per milliliter LPS was added for indicated time periods to mature cells. HD7 cells, a CD4+ T-cell clone that recognizes a peptide derived from mouse IgG1 antibodies in HLA-DR0101/DQw1, were used as T-cell responders (1 (link), 3 (link), 26 (link)).
Percoll gradient centrifugation
Manufactured by Cytiva
Sourced in Sweden, United States
Percoll gradient centrifugation is a technique used for the separation and purification of cells, organelles, and other biological particles. It involves the creation of a density gradient using the colloidal silica-based medium Percoll, which allows for the separation of components based on their density during centrifugation.
Automatically generated - may contain errors
Lab products found in correlation
Lymphoprep gradient, by Axis-Shield (1 mentions)
Interleukin 4 (il 4), by Thermo Fisher Scientific (1 mentions)
Gm csf, by Thermo Fisher Scientific (1 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
Glutamine, by Lonza (1 mentions)
Penicillin streptomycin, by Lonza (1 mentions)
Fetal bovine serum (fbs), by Lonza (1 mentions)
3 protocols using percoll gradient centrifugation
1
Monocyte-Derived Dendritic Cell Generation
2
Detecting PSCA Expression in Prostate Cancer
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Blood samples of 20 mL were obtained from patients just before the RP operation. Samples were delivered to the laboratory within an hour and nucleated cell fractions were isolated from 5 mL whole blood samples using Percoll Gradient Centrifugation (Amersham Biosciences, Uppsala, Sweden). Then, total RNA, extracted from nucleated cells, was converted to cDNA by reverse transcriptase and analyzed by PCR and nested PCR, as described in previous reports [10 (link), 11 (link)]. After RT-PCR assay sensitivity in cultured LNCaP cells was completed, assay products were evaluated to determine PSCA positivity. A 2% agarose gel was used for electrophoresis, and an ultraviolet transilluminator (UV-T) was used for visualization. All 110 PSCA-positive samples were collected and stored at −70°C.
3
Cardiomyocyte Isolation and Culture
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After scalpel homogenization, the heart tissue was incubated with 0.25% (w/v) trypsin overnight at 4 °C, following a 0.1% w/v) collagenase treatment for 20 min at 37 °C. Cardiomyocytes were enriched by percoll gradient centrifugation (Amersham, USA) and plated at a density of 5×10 5 /ml in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% (v/v) fetal calf serum at 37 °C and 5% (v/v) CO 2 for 24 hours as described previously [13] . Then the cardiomyocytes were washed by balanced salt solution and the medium was replaced. The cardiomyocytes were then underwent 72-hour further incubation.
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