Ethylene diamine tetraacetic acid (edta)

Bone marrow (BM) cells were collected by flushing femoral shafts with cold RPMI supplemented with 2% bovine serum albumin (BSA, US Biological, Swampscott, MA, USA) and 2 mM EDTA (IBI Scientific, USA). After depleting red blood cells using lysis buffer (Sigma-Aldrich, St. Louis, Missouri, USA), the cells were incubated with anti-B220 microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany), and the B-cells were isolated using positive selection with a magnetic activated cell-sorting (MACS) system (Miltenyi Biotec, Bergisch Gladbach, Germany). Single-cell suspensions of B220⁺ B-cells from BM were incubated with fluorescently labeled antibodies specific for CD43, B220, IgM, and CD23 in staining buffer (PBS with 0.5% BSA) for 20 minutes at 4°C, and cells were incubated with DAPI to select live cells (DAPI⁻). The cells were washed, and the immature B-cells were isolated according to the expression of the following surface markers: B220⁺, CD43⁻ CD23⁻, IgM⁺, and DAPI⁻. Cell sorting was performed using a FACS Influx Sorter (BD Biosciences). The purity of the sorted cells ranged from 95% to 98%.

Bidistilled water, ethanol, 85% formic acid, ethyl acetate, glacial acetic acid, dimethyl sulfoxide (DMSO), acetonitrile RS for HPLC, punicalagin (≥98%), ellagic acid (≥95%), phosphate buffered saline (PBS), dithiothreitol (DTT), oxidized glutathione (GSSG) and eosin isothiocyanate were purchased from Sigma (Milan, Italy). EDTA (0.5 M solution pH 8.0) from IBI Scientific (Dubuque, Iowa).
Pomegranate fruits of cv. Mollar (Ml) were obtained from an Italian market. Some fruits of Italian origin, belonging to two sub-varieties of cv. Dente di Cavallo (DC₁ and DC₂) with a difference of about two weeks in the maturation stage and harvest time, were provided by a local supplier (Azienda Biologica Giovomel, Avellino, Italy). A pomegranate variety from Mozia island (South Italy) was provided by the “Missione Archeologica Mozia” of “Sapienza” University of Rome (Mz).

Nine-week-old mice were euthanized, and BM cells were collected through flushing the femoral shafts with cold RPMI supplemented with 2% FBS and 2 mM EDTA (IBI Scientific, Dubuque, IA, USA). Red blood cells were depleted with a lysis buffer (Sigma-Aldrich, St. Louis, MO, USA) and incubated with anti-CD23 MicroBeads; mature recirculating B cells were removed with the magnetically activated cell-sorting (MACS) system (Miltenyi Biotec) through positive selection using LS columns (Miltenyi Biotec). The negative fraction was recovered and subsequently incubated with anti-B220 MicroBeads (Miltenyi Biotec), so that B220+CD23- BM cells were purified by positive selection.