Ficoll paque plus medium

Spleens from individual mice were harvested, and single-cell suspensions were prepared by meshing the mouse spleens through a 70-μm-mesh-size cell suspension mesh (BD), followed by centrifugation on Ficoll-Paque Plus medium (GE Healthcare) for 20 min at 600 × g at room temperature. Cells were collected and washed once with sterile fluorescence-activated cell sorting buffer (2% fetal calf serum [FCS] and 5 mM EDTA in PBS). Splenocytes (10⁶) were stained with the following antibodies: LIVE/DEAD fixable viability dye (Thermo Fisher), anti-mouse CD4-fluorescein isothiocyanate, anti-mouse CXCR5-phycoerythrin (PE), anti-mouse CD279-allophycocyanin (APC), anti-mouse TCRβ chain-BV510, anti-mouse CD19-APC, anti-mouse CD38-peridinin chlorophyll protein-Cy5.5, anti-mouse CD95-PE-Cy7, or isotype control antibodies. All antibodies were purchased from BD Pharmingen. Data were collected on a BD FACSAria flow cytometer (BD Biosciences) and analyzed with FlowJo software (TreeStar Inc., Ashland, OR, USA). A total of 50,000 cells were collected for each sample.

We isolated peripheral blood mononuclear cells (PBMCs) from the buffy coats of the 20 subjects by density gradient centrifugation using Ficoll-Paque PLUS medium (GE Healthcare Life Sciences, Pittsburgh, PA). We isolated monocytes from the PBMCs by positive selection using magnetic CD14 MicroBeads according to the supplier’s protocol (Miltenyi Biotec, San Diego, CA). We cultured isolated monocytes (1 × 10⁶ cells/mL) in RPMI 1640 medium (Gibco, Life Technologies, Grand Island, NY), 25 mg/mL Gentamicin (Gibco), and 10% charcoal-stripped fetal bovine serum (Gibco) in 24-well plates. Monocytes were cultured in three replicates for 24 hr for each of the following treatments: 1) vehicle solution containing 1% ethanol and 99% culture medium, as a negative control; 2) 100 nM of 1,25D; 3) 10 ng/mL of LPS in the vehicle solution; and 4) 100 nM of 1,25D and 10 ng/mL of LPS (experimental design summarized in Supplemental Material, Figure S1). These four treatments are abbreviated as E, V, L, and V + L, respectively.

For mRNA-seq, PBMCs were isolated from total blood using Ficoll-Paque™ PLUS medium (GE Healthcare, Uppsala, Sweden) from patients and volunteers. Total RNA was extracted from PBMCs using TRIzol reagent (Ambion, Carlsbad, CA). Sequencing libraries were prepared according to the manufacturer’s instructions (TruSeq® RNA LT Sample Prep Kit v2, Illumina, San Diego, CA), including purifying and fragmenting mRNA, synthesizing first-strand cDNAs, synthesizing second-strand cDNAs, performing end repair by adenylating the 3’ ends, ligating adapters, and enriching DNA fragments. The pooled library consisted of sequences with lengths of approximately 250 nucleotides. The library was sequenced using the HiSeq 2500 sequencing platform (Illumina, USA) as highlighted in the Additional file 2: Methods [18 (link)–21 (link)].

Approximately 15–25 ml of venous blood was drawn from each study subject into heparinized tubes and centrifuged at 2000 rpm for 10 min at 4℃ to extract plasma, which was stored at − 80℃. PBMCs were isolated from heparinized blood by density-gradient centrifugation using Ficoll-Paque Plus medium (GE Healthcare) in accordance with the manufacturer’s instructions.

The Pan Monocyte Isolation Kit was purchased from Miltenyi Biotec, Bergisch Gladbach, Germany. Lipopolysaccharide (LPS) from E. coli (055:B5), RPMI-1640 medium, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), human AB serum, ethylenediaminetetraacetic acid disodium salt (EDTA), penicillin/streptomycin, and accutase were from Sigma Aldrich (St. Louis, MO). Ficoll-Paque PLUS medium was purchased from GE Healthcare (Uppsala, Sweden), MEM (minimal essential medium) α was from Thermo Fisher Scientific (Waltham, MA), human platelet lysate from PL BioScience (Aachen, Germany), gentamycin from Lonza (Basel, Switzerland), and heparin from Ratiopharm (Ulm, Germany). Dulbecco’s phosphate buffered saline (DPBS) with (+/+) or without (−/−) calcium and magnesium was obtained from Life Technologies (Paisley, UK), and annexin V (Anx5) binding buffer was purchased from BD Biosciences (San Jose, CA).