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Extraction and culture of bone marrow macrophages was performed as previously described [39 (link)]. Bone marrow was extracted from tibias and femurs of C57BL/6 female mice (Jackson Lab) older than 8 weeks. After isolating both bones from both legs, bone marrow was flushed out of each cut bone using 23Gx3/4 needle (BD). Red blood cells were lysed in 0.83% freshly prepared NH₄Cl (Sigma) for 3 min and the remaining cells were seeded in 100 mm non-tissue culture treated plates (Corning) at a concentration of 3E+6 cells per plates in 8 ml of Dulbecco’s modified eagle medium with high glucose (DMEM; Corning) containing 20% (vol/vol) of L929 supernatant (LCM), 10% (vol/vol) of fetal bovine serum (FBS; Premium Select from R&D Systems), 10mM of HEPES (Sigma), 1mM of sodium pyruvate (Sigma), 0.05mM of β-mercaptoethanol (Sigma) and 100 U/ml of penicillin/streptomycin (Genesee Scientific). After 3 days, 10 ml of fresh medium was supplemented and the differentiated bone marrow derived macrophages were harvested 4 days later, on day 7. Macrophages were seeded in DMEM media supplemented with FBS, HEPES, sodium pyruvate and β-mercaptoethanol but without LCM or antibiotics in 6-well tissue culture treated plates at the concentration of 1E+6 macrophages per well if freshly harvested or 1.2E+6 macrophages per well if from frozen stock, to be infected the next day.

HEK293T, U2OS, and MDA-MB-231 cells were purchased from the American Type Culture Collection (ATCC CRL-3216, HTB-26, and HTB-96, respectively; Manassas, VA, USA) within the previous 24 months and passaged < 10 times for all experiments (passage numbers from 6–15). HEK293T cells were grown in DMEM high glucose plus L-Glutamine supplemented with 1% sodium pyruvate (Hyclone, Logan, UT, USA, #SH30022.01) and 10% fetal bovine serum (FBS, Premium Select, R&D systems, Minneapolis, MN, USA). U2OS cells were grown in RPMI 1640 with L-glutamine (Corning, Glendale, AZ, USA, 10-040-CV) supplemented with 10% FBS. MDA-MB-231 cells were grown in DMEM high glucose plus Glutamax (Life Technologies, Carlsbad, CA, USA, #10566016) supplemented with 1% sodium pyruvate and 10% FBS. Biweekly testing for mycoplasma contamination was performed using the Lonza MycoAlert (Lonza, Bend, OR, USA, #LT07-318).
Low-glucose-adapted HEK293T and MDA-MB-231 cells were grown in DMEM low glucose and pyruvate (Thermo Fisher Scientific, Waltham, MA, USA, #11885084) supplemented with L-glutamine, 1% sodium pyruvate, and 10% FBS. Low-glucose-adapted U2OS cells were grown in RPMI 1640 with L-glutamine without glucose (Life Technologies #11879020), supplemented with 5mM glucose (D-(+)-Glucose Solution Sigma, St. Louis, MO, USA, #G8644) and 10% FBS.

MDA-MB-231(MDA-231), MDA-MB-468 (MDA-468), and HEK293T were purchased from the American Type Culture Collection (ATCC HTB-26, HTB-132, and CRL-3216, respectively; Manassas, VA, USA) within the last 24 months and passaged < 15 times for all experiments. Cells were tested biweekly during experiments for mycoplasma contamination using the Lonza MycoAlert^® (Lonza #LT07-318). MDA-231 and MDA-468 cells were grown in DMEM High Glucose + GlutaMAX™ (Life Technologies, Carlsbad, CA, USA, #10566016) and supplemented with 1% sodium pyruvate (Life Technologies, #11360070) and 10% FBS (Premium Select, R&D systems, Minneapolis, MN, USA). HEK293T cells were grown in DMEM High Glucose + L-Glutamine (HyClone, Logan, UT, USA, # SH30022.01) and supplemented with 1% sodium pyruvate (Life Technologies #11360070) and 10% FBS. Cells were maintained in a humidified 37 °C incubator with 5% carbon dioxide.