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MB Sample ID: SA186791
| Local Sample ID: | NecJ 8h 3 |
| Subject ID: | SU002077 |
| Subject Type: | Cultured cells |
| Subject Species: | Mus musculus |
| Taxonomy ID: | 10090 |
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Collection:
| Collection ID: | CO002070 |
| Collection Summary: | Experimental animals This study was approved and performed in accordance with the ethical guidelines of the Institutional Animal Care and Use Committee at National Jewish Health. C57BL/6J (000664), B6-CD45.1 (002014) and CCR2KO (004999) mice were obtained from Jackson Laboratories (Bar Harbor, ME, USA). DsRed.T3 mice (available as 006051 at Jackson Labs) were generously shared by the Nagy laboratory (Vintersten et al., 2004). CCR2KO mice were crossed with DsRed mice until double homozygous. Mice used in experiments were between 8-16 weeks of age. Both male and female sex were used in experiments except bone marrow chimera experiments, which used only male mice. Cell Lines Jurkat (human T lymphocytes, clone E6-1) were obtained from ATCC and cultured in RPMI-1640 (GIBCO) supplemented with 10% heat-inactivated fetal bovine serum (v/v) (GeminiBio), 1X Pen/Strep/Glut, 1mM HEPES, and 100M Sodium Pyruvate. Cells were cultured in a humidified CO2 incubator at 37C. The Jurkat cell line was established from peripheral blood of a 14-year-old male diagnosed with T cell leukemia. Murine primary cell cultures 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 100M Sodium Pyruvate) plated 1.3x106 macrophages/well in 6-well plates for FACS sorting or 4x106 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, 100M Sodium Pyruvate, 55M beta-Mercaptoethanol). All macrophages were removed from culture dishes by 3-minute incubation with 1X Trypsin-EDTA (Sigma) plus gentle scraping. |
| Sample Type: | Macrophages |
