Magnetic-activated cell sorting (MACS, Miltenyi) was performed to positively separate CD14+ monocytes from 20 million PBMCs, as per the manufacturer’s instructions. This is designed to provide a sample of ~99% purity. Flow cytometry was performed on a subset of samples with similar purities observed. Typical monocyte yields ranged from 1 × 106 to 10 × 106 monocytes per individual, of which 5 × 105 untreated cells were immediately suspended in RLT reagent (Qiagen) to form the naïve subset and snap-frozen for RNA extraction at a later date. All other monocytes were resuspended at a concentration of 1 × 106/ml in 400 μl of prewarmed RPMI 1640 complete medium, supplemented with 20% fetal calf serum, penicillin/streptomycin, and l -glutamine. Cells were rested overnight (16 hours) at 37°C, 5% CO2 in 5-ml nonadherent polypropylene cell culture tubes (BD Biosciences) before stimulation assays.
Nonadherent polypropylene cell culture tubes
Manufactured by BD
Nonadherent polypropylene cell culture tubes are designed for the in vitro cultivation of cells. They are made of polypropylene, a non-adherent material that prevents cell attachment to the tube surface, allowing for the maintenance of cells in suspension. These tubes are suitable for a variety of cell culture applications.
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Lab products found in correlation
Rlt reagent, by Qiagen (1 mentions)
Magnetic activated cell sorting (macs), by Miltenyi Biotec (1 mentions)
Positive selection, by Miltenyi Biotec (1 mentions)
Tumor necrosis factor (tnf), by R&D Systems (1 mentions)
Interleukin 7 (il 7), by Thermo Fisher Scientific (1 mentions)
Pam3cysk4, by InvivoGen (1 mentions)
Imiquimod, by InvivoGen (1 mentions)
Lipopolysaccharides (lps), by InvivoGen (1 mentions)
2 protocols using nonadherent polypropylene cell culture tubes
1
Purification and Stimulation of Monocytes
2
Isolation and Stimulation of Monocytes
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Whole blood was collected into EDTA tubes (BD vacutainer system) and peripheral blood mononuclear cells were obtained by density centrifugation (Ficoll Paque). CD14 cell isolation was carried out by positive selection (Miltenyi) according to the manufacturer’s instructions. Cells were rested overnight (16 h) at 37 °C, 5% CO2 in 5 ml non-adherent polypropylene cell-culture tubes (BD Biosciences) prior to stimulation assays. Cells were stimulated for 24 h in RPMI1640 media with 20% fetal calf serum in the presence of 20 ng/ml LPS (Invivogen), 100 ng/ml pam 3cysk4 (Invivogen), 100 ng/ml imiquimod (Invivogen), 10 ng/ml TNF (R&D systems), 10 ng/ml IL-7 (Peprotech) and CD2/3/28 beads (Miltenyi) at a ratio of 1 bead to 2 cells. In all experiments an unstimulated incubator control was included. TNF blockade was achieved with 5 µg/ml of infliximab (Remicade, Janssen). Macrophages were differentiated in the presence of M-CSF (50 ng/ml) as per previously described25 (link).
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