Hybrimax dmso

PBMCs from subjects were obtained by leukapheresis and isolated by density gradient centrifugation (Lymphocyte Separation Medium; Wisent, St-Bruno, QC) and cryopreserved in 10% dimethyl sulfoxide (Hybri-Max DMSO; Sigma-Aldrich, St Louis, MO); 90% Heat-Inactivated Fetal Bovine Serum (HI-FBS) (PAA Laboratories, Etobicoke, ON).

Suberoylanilide hydroxamic acid (SAHA) (Sigma-Aldrich), prostratin (Sigma-Aldrich), romidepsin (Selleckchem), panobinostat (Selleckchem), and hexamethylene bisacetamide (HMBA) (Sigma-Aldrich) were dissolved in hybrimax DMSO (Sigma-Aldrich) at the indicated concentrations. IL-7, IL-15, and IL-2 were purchased from R&D Systems and dissolved in sterile PBS. IL-15SA was generated by dissolving IL-15 and IL-15Rα-Fc (R&D Systems) in sterile PBS and combining these in equimolar ratios. Stocks of the above reagents were flash-frozen in single-use aliquots in EtOH dry-ice baths. ALT-803 was obtained from Altor Bioscience Corporation and stored at 4°C.

B cells were isolated from spleen of at least 8-week-old mice using the Miltenyi Biotec murine B-cell isolation kit. Following isolation, cells were counted and either immediately plated in triplicate wells in complete B-cell medium (DMEM:H12 with 10% Hyclone FBS, L-glutamine, penicillin/streptomycin, 12 mM HEPES, and 55 μM Beta-mercaptoethanol) at 2 × 10⁶ cells/mL, or stained with CFSE (Thermo Fisher) before plating. B cells were stimulated with either 5 or 10 μg/mL of goat anti-mouse IgM F(ab)2 (Sigma), 2 or 20 μg/mL K12 LPS (Invivogen, San Diego CA), 100 or 500 ng/mL recombinant human BAFF (Peprotech, Rocky Hill NJ), and were sometimes supplemented with 20 ng/mL of murine IL-4 (Peprotech) or 1 μg/mL LEAF-purified anti-mouse CD40 antibody RRID:AB_312942(Biolegend). For MG132 experiments, cells were incubated with either Hybrimax DMSO (Sigma Aldrich) or 5 μM of MG132 (EMD Millipore, Billerica MA).

The National Center for Cell Science (NCCS), located in Pune, Maharashtra, India, provided the breast cancer cell lines MCF-7, MDA-MB-231, and MDA-MB-468. Prof. Annapoorni Rangarajan from the Department of Molecular, Reproduction, Development and Genetics at the Indian Institute of Science in Bengaluru, Karnataka, India, provided the triple positive breast cancer cell line BT-474. Dr. Gopinath, M.S. Principal Scientist, Department of Molecular Nutrition, CSIR-CFTRI, Mysore, Karnataka, India, kindly supported by providing the human keratinocyte cell line HaCaT. Cell culture grade Hybrimax DMSO, SRB, DPPH, Trolox, and TPTZ were acquired from Sigma Chemical Company in St. Louis, Missouri, in the United States. The following products were purchased from HiMedia Laboratories in Mumbai, Maharashtra, India: DMEM, FBS, PenStrep, Glutamax, Trypsin, and DPBS. For cell culture, Techno Plastic Products India, Pvt. Ltd. is a company in Bengaluru, Karnataka, India provided plastic ware (T25, T75, 15.0 mL and 50.0 mL conical tubes, disposable pipettes, micropipettes tips) etc. All other reagents, including solvents, salts, acids, and bases, were of Analytical Reagent (AR) grade, purchased from Sisco Research Laboratories Pvt. Ltd. (SRL), Mumbai, Maharashtra, India.

HL60 (ATCC) cells were grown to 5 × 10⁵ cells/ml in complete medium (RPMI, 25 mM HEPES, l-glutamine (Gibco), 10% heat-inactivated FBS (Sigma), 1% penicillin/streptomycin (Corning/cellgro). Cells were differentiated in 1.3% Hybri-Max DMSO (Sigma) for 6–7 days at a concentration of 1 × 10⁵ cells/ml. Cells were untreated (control), treated with etomoxir (10 μM), octanoic acid (100 μM) with etomoxir (10 μM) or octanoic acid (100 μM) alone for 2 h at 37 °C. Cells were pelleted and washed once with PBS and used for specific assays.

Aβ42 oligomers were prepared as described previously (Dahlgren et al, 2002 (link)). Briefly, Aβ42 peptide was dissolved in hexafluoroisopropanol (FUJIFILM Wako Pure Chemicals Corp., Osaka, Japan) to a concentration of 1 mM. After removing hexafluoroisopropanol under vacuum with a SpeedVac system, the peptide was resuspended in dimethyl sulfoxide Hybri‐Max (DMSO) (Sigma‐Aldrich, St. Louis, MO, USA) to a concentration of 5 mM. Ham's F‐12 (phenol red‐free, FUJIFILM Wako Pure Chemicals Corp.) was then added to adjust the final concentration of the peptide to 100 μM. The peptide was then incubated at 4°C for 24 h. Stock solutions (5 mM) of p3‐Alcα and p3‐Alcβ peptides were prepared by dissolving each in DMSO. For the detection of Aβ42, p3‐Alcβ37, and p3‐Alcα35 by immunoblotting, the respective DMSO‐containing peptide solutions were diluted in PBS to 10 μM and incubated at 37°C for 24 h. The peptide solutions were then centrifuged at 20,400 g for 10 min at 4°C and the resultant supernatants analyzed with Tris–Tricine polyacrylamide gel SDS electrophoresis followed by immunoblotting with the indicated antibodies.