Peripheral blood mononuclear cells (PBMCs) were isolated using lymphocyte separation medium (Ficoll-Hypaque density gradient, Axis-shield, Germany) from 10 ml fresh heparinized blood sample. And then PBMCs were incubated with 1 μM CpG-B ODN2006 (In vivoGen, San Diego, CA, USA) for 96 h at 37°C. PMA (3 ng/ml) and ionomycin (100 ng/ml) were added during the last 4h in the presence of 10 μg/ml brefeldin A (all from Sigma, St Louis, MO, USA). Cells were then surface stained for the markers CD19 Pacific Blue-A, and stained intracellularly with anti-IL-10 APC. So we could assess the ability and the frequency of IL-10 production from purified B cells (14 (link)). All antibodies including CD19-PE and IL-10-APC used in flow cytometry were ordered from BD Pharmingen (BD Pharmingen, San Diego, CA, USA). PBMCs were acquired on a FACS Canton flow cytometer (Becton-Dickinson, Franklin Lakes, NJ, USA). FlowJo 7.6.1 (Tree Star Inc., Ashland, OR, USA) was used as the flow analysis software.
Ficoll hypaque density gradient
Manufactured by Axis-Shield
Sourced in Germany, Norway
Ficoll-Hypaque density gradient is a laboratory product used for cell separation and purification. It is a mixture of Ficoll, a synthetic high-molecular-weight polymer, and sodium diatrizoate, a radiographic contrast agent. The product creates a density gradient that allows the separation of different cell types based on their buoyant density when centrifuged.
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2 protocols using ficoll hypaque density gradient
1
Assessing IL-10 Production in B Cells
2
PBMC Isolation and Preservation
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Peripheral venous blood samples were collected from all subjects into 5 mL tubes, containing EDTA, as the anticoagulant. Within several hours from the collection, PBMCs were separated from the samples, via Ficoll-Hypaque density gradient centrifugation (Axis-Shield PoC AS, Oslo, Norway). Approximately 5 × 106 PBMCs were collected from each sample, processed with 1 mL of TRIzol (Invitrogen, Carlsbad, CA, USA) and stored at -80°C, until analyzed.
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