Alpha minimal essential medium α mem

Recombinant human RANKL and human macrophage colony-stimulating factor (M-CSF) were purchased from PeproTech EC, Ltd. (London, UK). The β-actin antibody was obtained from Sigma-Aldrich (St. Louis, MO, USA). Specific antibodies against phospho-p38, p38, phospho-ERK 1/2, ERK 1/2, phospho-JNK, JNK, phospho-IκB, phospho-AKT, and AKT, c-FOS, nuclear factor of activated T cells c1 (NFATC1), and IκB were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Alpha minimal essential medium (α-MEM), fetal bovine serum (FBS), and penicillin-streptomycin solution were purchased from Gibco BRL (Grand Island, NY, USA).

Two E. faecalis isolated strains from retreated root canal samples and E. faecalis OG1RF were used in this study. The two isolated strains are called CA1 (CRISPR-Cas absence strain 1) and CA2 (CRISPR-Cas absence strain 2) due to their lack of a CRISPR-Cas module (Tong et al., 2017 (link)). We selected the E. faecalis OG1RF as a standard plasmid-free strain presenting the intrinsic CRISPR loci. Following routine streaking on brain heart infusion (BHI) agar (Difco, Becton Dickinson, Sparks, MD, USA) and aerobic culture, we inoculated a single bacterial colony into BHI broth to allow the bacteria to reach the exponential growth phase.
We cultured RAW264.7 murine macrophage line (ATCC, Manassas, VA) cells in alpha-minimal essential medium (α-MEM; Gibco, New York, NY, USA) with 10% fetal bovine serum (FBS; Gibco, New York, NY, USA) in a 5% CO₂ humidified incubator at 37°C.

Dulbecco's modified eagle's medium (DMEM) was purchased from Welgene (Daejeon, Korea). An alpha-minimal essential medium (α-MEM), penicillin-streptomycin solution, and fetal bovine serum (FBS) were purchased from Gibco (Grand Island, NY); recombinant mouse RANKL was purchased from Peprotehc (London, UK); an osteo assay surface plate was obtained from Corning, Inc. (Corning, NY); a cell titer 96 aqueous nonradioactive cell proliferation assay kit was obtained from Promega (Madison, WI); antiactin and anti-c-fos were obtained from Santa Cruz Biotechnology (Santa Cruz, CA); anti-NFATc1 was purchased from BD pharmingen (San Diego, CA); a reverse-transcription kit was purchased from Invitrogen (Carlsbad, CA); and the taq polymerase was purchased from Kapa Biosystem (Wilmington, MA). All primer pairs were made by Genotech (Daejeon, Korea). In addition, the TRAP staining kit, 17b-estradiol (E2), and kaempferol were purchased from Sigma-Aldrich (St. Louis, MO); isoflurane was purchased from Hana Pharm (Hwasung, Korea); and pentobarbital sodium was obtained from Hanlim Pharm (YoungIn, Korea).

KCl was obtained from ACP Inc (Montreal, QC, Canada) and Na₂SO₄ from Laboratoire MAT (Québec city, QC, Canada). Formic acid, 1.0 M HCl, and 1.0 M NaOH solutions were from Fisher Scientific (Montreal, QC, Canada), trifluoroacetic acid was purchased from J.T. Baker (Phillipsburg, NJ, USA). NaCl, Acetonitrile optima^® liquid chromatography-mass spectrometry (LC/MS), and water grade were from VWR international (Montréal, QC, Canada). Concerning the glucose uptake experiments, the alpha-Minimal Essential Medium (α-MEM), Fetal Bovine Serum (FBS), and trypsin (0,25% solution) were obtained from Invitrogen (Burlington, ON, Canada). The 2-déoxy-D-glucose (non-radioactive), CaCl₂, Hepes-Na, and MgSO4 were purchased from Sigma Aldrich (Oakville, ON, Canada). D-2-deoxy-[³H] glucose was from Perkin Elmer (Woodbridge, ON, Canada) and Pierce^® BCA Protein Assay Kit BCA was from Pierce Biotechnology (Rockford, IL, USA). Insulin was from CHUL’s pharmacy (Québec, QC, Canada). Also, the L6 skeletal muscle cells line, derived from neonatal rat thigh skeletal muscle, were provided by Dr. A. Klip, Hospital for Sick Children (Toronto, ON, Canada).

Alpha-Minimal Essential Medium (α-MEM), Dulbecco's modified Eagle's medium F12, and Earle's Balanced Salt Solution cell culture media were obtained from Invitrogen. Fetal calf serum was purchased from Internegocios S.A. Kanamycin (50 μg/ml), chloramphenicol (20 μg/ml), and ampicillin (100 μg/ml); MTT, firefly luciferase, suramin, PPADS, 8-phenyl theophylline, NF110, HK, apyrase, ATP, ADP, AMP, UTP, and suramin were purchased from Sigma-Aldrich. LIVE/DEAD BacLight Bacterial Viability kit and 4',6-diamidino-2-phenylindole were purchased from Molecular Probes.