Monocytes were sorted from PBMCs by using CD14-coated microbeads (Miltenyi Biotech, Paris, France). Cells, preincubated or not with diphenyleneiodonium (DPI) or N-acetylcysteine (NAC) for 3 hours at 37°C were washed twice and cocultured in 1-μm–pore size inserts with BJ cells (fibroblasts established from skin [ATCC CRL-2522]) placed on coverslips in 24-well companion plates. PBMCs or monocytes and BJ cells were cocultured in a 2:1 ratio in 1:1 Dulbecco modified Eagle medium and RPMI culture medium supplemented with 10% heat-inactivated FBS for 3 days. Camptothecin (10 μM) was used on the BJ cells for 45 minutes at 37°C. LPS (1 μg/mL) or angiotensin II (AngII) (75 pM) was added to the cells in a 500-μL final volume of RPMI culture medium without serum and incubated at 37°C for 30 minutes. The cells were washed and fixed for further staining.
Cd14 coated microbeads
Manufactured by Miltenyi Biotec
Sourced in France
CD14-coated microbeads are a type of immunomagnetic separation tool used for the isolation and purification of CD14-positive cells from complex biological samples. The microbeads are coated with antibodies specific to the CD14 surface marker, allowing for the efficient capture and separation of CD14-expressing cells.
Automatically generated - may contain errors
Lab products found in correlation
Pyruvate, by Thermo Fisher Scientific (1 mentions)
Paxgene tube, by Preanalytix (1 mentions)
Ficoll paque density gradient centrifugation, by GE Healthcare (1 mentions)
Glutamine, by Thermo Fisher Scientific (1 mentions)
96 well flat bottom plate, by Corning (1 mentions)
Rpmi 1640 dutch modified culture medium, by Thermo Fisher Scientific (1 mentions)
Lipopolysaccharides (lps), by Merck Group (1 mentions)
2 protocols using cd14 coated microbeads
1
Monocyte-Fibroblast Coculture Assay for Oxidative Stress
2
Isolation and Stimulation of Human Monocytes
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EDTA plasma was obtained by venous blood sampling and was stored using standardized protocols. Plasma total cholesterol, HDLc and LDL-c levels were analyzed using commercially available enzymatic methods. PAXgene tubes (Preanalytix, GmbH, Switzerland) were incubated for at least 2 h at room temperature before storage at À20 C until RNA isolation.
Human peripheral blood mononuclear cells (PBMCs) were isolated from EDTA blood using Ficoll-Paque density gradient centrifugation (GE Healthcare, UK). Monocytes were isolated by magnetic activated cell sorting (MACS) using CD14-coated MicroBeads according to manufacturer's instructions (MACS, Miltenyi Biotec, Leiden, The Netherlands). Monocytes were diluted to 1 Â 10 6 /ml in RPMI 1640 Dutch-modified culture medium (Life Technologies/ Invitrogen, Breda, The Netherlands) supplemented with 10 mM glutamine (Invitrogen), 10 mg/mL gentamicin (Centraform), 10 mM pyruvate (Invitrogen). A total of 5 Â 10 5 monocytes were seeded per well on flat-bottom 96-well plates (Corning, New York, USA) and stimulated for 24 h with RPMI only as a negative control, 10 ng/ml LPS (Sigma-Aldrich, St. Louis, MO; from E. coli serotype 055:B5, further purified as described [7] ) or 10 mg/ml Pam3Cys (EMC microcollections, Tübingen, Germany; L2000). After 24 h, plates were centrifuged and supernatants were stored at À20 C until cytokine assessment.
Human peripheral blood mononuclear cells (PBMCs) were isolated from EDTA blood using Ficoll-Paque density gradient centrifugation (GE Healthcare, UK). Monocytes were isolated by magnetic activated cell sorting (MACS) using CD14-coated MicroBeads according to manufacturer's instructions (MACS, Miltenyi Biotec, Leiden, The Netherlands). Monocytes were diluted to 1 Â 10 6 /ml in RPMI 1640 Dutch-modified culture medium (Life Technologies/ Invitrogen, Breda, The Netherlands) supplemented with 10 mM glutamine (Invitrogen), 10 mg/mL gentamicin (Centraform), 10 mM pyruvate (Invitrogen). A total of 5 Â 10 5 monocytes were seeded per well on flat-bottom 96-well plates (Corning, New York, USA) and stimulated for 24 h with RPMI only as a negative control, 10 ng/ml LPS (Sigma-Aldrich, St. Louis, MO; from E. coli serotype 055:B5, further purified as described [7] ) or 10 mg/ml Pam3Cys (EMC microcollections, Tübingen, Germany; L2000). After 24 h, plates were centrifuged and supernatants were stored at À20 C until cytokine assessment.
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