Mice on C57BL/6 genetic background were housed in a specific pathogen-free (SPF) facility on the basis of standard humane animal husbandry protocols, which were approved by the animal care and use committee of the Institute of Microbiology (Chinese Academy of Sciences). Bone marrow-derived macrophages (BMDMs) were collected from tibiae and femurs of 7-8 weeks old mice. After lysis of red blood cells, BMDMs were cultured in DMEM supplemented with 10% FBS, 1% Penicillin-Streptomycin Solution (Caisson) and Murine M-CSF (Pepro Tech) for 4–6 days. Bone marrow mesenchymal stem cells (BMSCs) were also collected from tibiae and femurs of 7-8 weeks old mice and cultured following a published protocol82 (link), and were used for experiments at the fourth passage.
Penicillin streptomycin solution
Manufactured by Caisson
Sourced in United States
Penicillin-Streptomycin Solution is a sterile liquid media supplement used in cell culture applications. It contains the antibiotics penicillin and streptomycin, which inhibit the growth of contaminating bacteria and fungi.
Automatically generated - may contain errors
Lab products found in correlation
Murine m csf, by Thermo Fisher Scientific (2 mentions)
Ovcar3, by American Type Culture Collection (1 mentions)
Skov3, by American Type Culture Collection (1 mentions)
Mesenchymal stem cell growth kit, by American Type Culture Collection (1 mentions)
A2780, by Merck Group (1 mentions)
A2780cis, by Merck Group (1 mentions)
B6.129 tlr2tm1kir j, by Jackson ImmunoResearch (1 mentions)
Mabg mtlr2, by InvivoGen (1 mentions)
Doramapimod, by Cayman Chemical (1 mentions)
L glutamine 200 mm, by Merck Group (1 mentions)
Hyclone, by Thermo Fisher Scientific (1 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
5 protocols using penicillin streptomycin solution
1
Isolation and Culture of Murine Immune Cells
2
Authentication and Cultivation of Ovarian Cancer Cell Lines
3
Isolation and Culture of Murine BMDMs
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Bone marrow-derived macrophages (BMDMs) were collected from tibiae and femurs of 8–12-weeks old mice. After lysis of red blood cells, BMDMs were cultured in DMEM supplemented with 10% FBS, 1% Penicillin-Streptomycin Solution (Caisson) and Murine M-CSF (Pepro Tech) for 4–6 days.
4
Macrophage Activation Assay with TLR2
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Male dahl-S rats (S), male C57BL/6J mice, or male Tlr2 knockout mice (B6.129-Tlr2tm1Kir/J; Jackson Laboratory, Bar Harbor, ME, USA) were injected intraperitoneally with thioglycolate, as previously described [31 (link)]. After 72 h, peritoneal macrophages were obtained by lavage and adherent macrophages were allowed to settle for another 72 h. Cells were allowed to grow in 12-well plates and were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Catalog No. 11995065; ThermoFisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (Rocky Mountain Biologicals, Inc, Missoula, MT, USA) and 1% penicillin–streptomycin solution (Caisson Labs, Smithfield, UT, USA).
MC-LR was used at a dose of 10 μM. Anti-Tlr2 monoclonal Ab (Item No. mabg-mtlr2; InvivoGen, San Diego, CA, USA), used at a dose of 2.5 μg/mL, with pretreatments for 1 h. Doramapimod (Item No. 10460; Cayman Chemical, Ann Arbor, MI, USA), used at a dose of 10 μM, with pretreatments for 30 min. All treatments were for 24 h in duration. Treatments were preceded by 24 h of serum starvation using DMEM supplemented with only 1% penicillin–streptomycin solution, and treatments were prepared in the same serum-starved conditions.
MC-LR was used at a dose of 10 μM. Anti-Tlr2 monoclonal Ab (Item No. mabg-mtlr2; InvivoGen, San Diego, CA, USA), used at a dose of 2.5 μg/mL, with pretreatments for 1 h. Doramapimod (Item No. 10460; Cayman Chemical, Ann Arbor, MI, USA), used at a dose of 10 μM, with pretreatments for 30 min. All treatments were for 24 h in duration. Treatments were preceded by 24 h of serum starvation using DMEM supplemented with only 1% penicillin–streptomycin solution, and treatments were prepared in the same serum-starved conditions.
5
4T1 murine breast cancer cell culture
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Cell culture and chemicals. 4T1 murine breast carcinoma cells with stably transfected multi-cistronic reporter genes have been reported previously, so called 4T1_PB3R (24) . Cells were cultured in RPMI1640 medium (Gibco; Invitrogen Inc., Carlsbad, CA, USA) containing 10% fetal bovine serum (FBS; HyClone; Thermo, Waltham, MA, USA), 1% penicillin-streptomycin solution (Caisson Laboratories Inc., North Logan, UT, USA) and 1% l-glutamine (200 mM) (Sigma-Aldrich Co., St. Louis, MO, USA). Cells were cultured in a 37˚C, humidified incubator containing 95% air and 5% CO 2 and passaged every 2 days. Preparation of glycated chitosan (10 mg/ml, dissolved in deionized distilled water) has been described previously (16) . It was then stored in the refrigerator until used.
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