Blood from healthy subjects was obtained from the Blood collection center of Angers (agreement ANG 2003-2). Human peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll-Paque (Amersham Biosciences, Uppsala, Sweden) density-gradient centrifugation. To generate Mϕ, monocytes were purified from PBMC by positive selection using anti-CD14 mAb-coated magnetic beads (Miltenyi Biotech, Bergisch Gladbach, Germany); purity was >99% (data not shown). Purified CD14+ monocytes (1 × 106 cells/ml) were differentiated into Mϕ by 5-day culture with 20 ng/ml M-CSF (Biotechne) and 2 ng/ml GM-CSF (CellGenix, Freiburg, Germany) in complete medium (CM), consisting of RPMI 1640 medium containing 2 mM l -glutamine, antibiotics (all from Lonza, Verviers, Belgium), and 10% (v/v) heat-inactivated fetal calf serum (Biowest, Nuaillé, France). After Ficoll-Paque centrifugation, neutrophils were isolated from erythrocytes by 1.5% (w/v) dextran (Amersham Biosciences) density-gradient sedimentation. Contaminating red blood cells were lysed by hypo-osmotic shock. Purity was routinely >98% (data not shown).
Heat inactivated fetal calf serum
Manufactured by Biowest
Sourced in Belgium
Heat-inactivated fetal calf serum is a component of cell culture media. It provides essential nutrients and growth factors to support the growth and proliferation of cells in vitro.
Automatically generated - may contain errors
Lab products found in correlation
Dextran, by Cytiva (1 mentions)
Gm csf, by CellGenix (1 mentions)
M csf, by Bio-Techne (1 mentions)
Ficoll paque, by Cytiva (1 mentions)
Anti cd14 mab coated magnetic beads, by Miltenyi Biotec (1 mentions)
Rpmi 1640 medium, by Lonza (1 mentions)
L glutamine, by Lonza (1 mentions)
Rankl, by Miltenyi Biotec (1 mentions)
M csf, by Miltenyi Biotec (1 mentions)
2 protocols using heat inactivated fetal calf serum
1
Isolation and Differentiation of Human Monocytes and Neutrophils
2
Osteoclast Differentiation from Bone Marrow
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Bone marrow cells were purified from long bones of 6-to 8-week-old C57BL/6 Dock5 wild-type and knockout mice sacrificed by cervical dislocation. 3.10 6 bone marrow cells were cultured for 3 days in αMEM containing 10% heat-inactivated fetal calf serum (Biowest), 2 mM glutamine and 100U/ml penicillin-streptomycin supplemented with 100 ng/ml M-CSF (Miltenyi, in 100mm-diameter petri dishes.
Medium was changed and the cells cultured for another 3 days to obtain BMMs. For osteoclast differentiation, 3.10 4 BMMs/well were seeded in 24-well tissue culture plates and cultured with 30 ng/ml M-CSF and 50 ng/ml RANKL (Miltenyi, #130-094-076) for 4-6 days. Media was changed every 2 days.
Medium was changed and the cells cultured for another 3 days to obtain BMMs. For osteoclast differentiation, 3.10 4 BMMs/well were seeded in 24-well tissue culture plates and cultured with 30 ng/ml M-CSF and 50 ng/ml RANKL (Miltenyi, #130-094-076) for 4-6 days. Media was changed every 2 days.
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