Peritoneal cells were isolated from naïve mice by lavage with 10mL of PBS containing 0.5mM EDTA. Lavage was estimated to contain 50% macrophages. Peritoneal macrophages were isolated by adhesion purification of lavage cells in RPMI-10 (RPMI-1640 containing 10% heat-inactivated fetal bovine serum (v/v), 1X Pen/Strep/Glut, 1mM HEPES, and 100μM Sodium Pyruvate) plated 1.3×106 macrophages/well in 6-well plates for FACS sorting or 4×106 macrophage/well in 24-well plates for flow cytometry. Non-adherent cells were removed by washing after 3–5 hours, and macrophages were cultured in fresh RPMI-10 overnight. 400ng/mL purified E. coli O111:B4 LPS (List Biological Laboratories Inc.) was added to generate inflammatory macrophages 12h before the addition of target cells. Bone marrow-derived macrophages were generated by 8-day culture of murine bone marrow cells grown in media containing M-CSF (High glucose DMEM containing 10% v/v FBS, 20% v/v L929-conditioned media, 1X Pen/Strep/Glut, 100μM Sodium Pyruvate, 55μM beta-Mercaptoethanol). All macrophages were removed from culture dishes by 3-minute incubation with 1X Trypsin-EDTA (Sigma) plus gentle scraping.
E coli o111 b4 lps
Manufactured by List Biological Laboratories
E. coli O111:B4 LPS is a lipopolysaccharide extracted from the cell wall of Escherichia coli O111:B4. It is a component of the outer membrane of Gram-negative bacteria and is commonly used in research applications.
Automatically generated - may contain errors
Lab products found in correlation
Trypsin edta, by Merck Group (4 mentions)
X vivo 10, by Lonza (2 mentions)
Taqman gene expression assay primer, by Thermo Fisher Scientific (2 mentions)
Sensifast probe hi rox one step kit, by Meridian Bioscience (2 mentions)
Rneasy micro isolation kit, by Qiagen (2 mentions)
Monocyte isolation kit 2, by Miltenyi Biotec (1 mentions)
7 protocols using e coli o111 b4 lps
1
Isolation and Activation of Murine Peritoneal Macrophages
2
Generating Inflammatory Macrophages from Human Monocytes
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Human monocyte-derived macrophages (HMDM) were generated from donor peripheral blood mononuclear cells (PBMC). PBMC were differentiated into HMDM by culture in X-Vivo10 (Lonza) containing 10% human serum (pooled from deidentified donors and heat inactivated, National Jewish Health Blood Prep Core) for 7 days. HMDM were plated at 1.3×106 macrophages/well in 6-well plates for FACS sorting. 400ng/mL purified E. coli O111:B4 LPS (List Biological Laboratories Inc.) was added to generate inflammatory macrophages 12h before the addition of target cells. HMDM were removed from culture dishes by 3-minute incubation with 1X Trypsin-EDTA (Sigma) plus gentle scraping.
3
Macrophage LPS Response Modulation
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Murine bone marrow-derived macrophages were plated and co-cultured with ApoJ for 1h. Following removal of unengulfed ApoJ by repeated washing with PBS, macrophages were treated with 400ng/mL purified E. coli O111:B4 LPS (List Biological Laboratories Inc.) ± 100μM NSC23766 for 6h, then RNA was collected and isolated using the RNeasy micro isolation kit (QIAGEN) per the manufacturer’s instructions. RT-PCR was performed using the SensiFAST Probe Hi-ROX One-Step Kit (Meridian Life Science Inc.) per manufacturer’s instructions with Taqman gene expression assay primers (Invitrogen). Expression of il-1b (Assay ID: Mm99999061_mH) and il-6 (Assay ID: Mm00446190_m1) was normalized to b2m (Assay ID: Mm00437762_m1).
4
Isolation and Stimulation of Monocytes
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Eight mL of whole blood was drawn from healthy subjects into BD vacutainer cell preparation tubes with sodium citrate (BD, Franklin Lakes, NJ) and centrifuged at 1500 RCF for 20 minutes. The PBMC layer was collected and washed twice with PBS, according to the manufacturer’s instructions. Monocytes were isolated from this preparation of PBMCs using the Miltenyi Biotec monocyte isolation kit II (Auburn, CA). With this kit, non-monocytes were indirectly magnetically labeled using a cocktail of biotin-conjugated antibodies and anti-biotin microbeads. Enriched monocytes were collected after running the preparation on LS columns, retaining all of the non-monocytes in the columns. This procedure was completed according to the manufacturer’s instructions. Isolated monocytes were plated in RPMI media plus 10% FBS in a 96 well plate, with 50,000 monocytes per well. Monocytes were stimulated with ultrapure E. coli O111:B4 LPS (List Biological Laboratories) for four hours.
5
Generating Inflammatory Macrophages from Human Monocytes
6
Macrophage LPS Response Modulation
7
Isolation and Activation of Murine Peritoneal Macrophages
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