Rpmi 1640 with glutamax

T cells and DCs were cultured in complete media consisting of RPMI 1640 with Glutamax (Life Technologies, Grand Island, NY, USA), supplemented with 5% heat inactivated human serum (Sigma, St. Louis, MO, USA). DC were cultured overnight with 500 U/mL each of recombinant IL-4 and GM-CSF (Peprotech, Rocky Hill, NJ, USA) and matured with 1000 U/mL recombinant IFN-γ (Peprotech) and 1 ng/mL LPS (Sigma). Prior to coculture with DC were tested for maturation status by CD83, CD86, and HLA-DR expression by flow cytometry and IL-12 production by ELISA (R&D Systems, Minneapolis, MN, USA). T cells were labeled with violet proliferation dye 450 (BD) according to the manufacturer’s instructions. T cells were cultured with DC at a 10:1 ratio, incubated at 37°C for the indicated timepoints, and analyzed for proliferation and activation by flow cytometry. Supernatants were collected and cytokines were measured by multiplex analyses (MesoScale Discovery, Rockville, MD, USA). For ELISPOT analysis, cells were collected on day 6 of MLR and analyzed for IFN-γ spot production using pre-coated plates (MabTech, Cincinnati, OH, USA). For intracellular detection of cytokines, cells were collected on day 6 of the MLR and treated with PMA (Sigma), ionomycin (Sigma), and GolgiPlug (BD, San Jose, CA, USA) for 6 h prior to addition of antibodies for flow analysis.

Human blood basophils and mouse splenocytes were cultured in a culture medium (RPMI 1640 with Glutamax and 20 mM HEPES, 1 mM Na-pyruvate, and 1× non-essential amino acids; all from Life Technologies, Carlsbad, CA, USA), 100 μg/ml streptomycin and 100 U/ml penicillin (GE Healthcare, Chicago, IL, USA), and 37.5 μM β-mercaptoethanol (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 20% heat-inactivated fetal calf serum (Life Technologies) at 37°C and 5% CO₂. For human basophils, 10,000 cells in 200 µl medium per point were used. For mouse splenocytes, 2 million cells in 200 µl medium per point were used. The cells were pretreated or not with 1 µM AMG853 (Tocris, Bio-Techne) over 15 min at 37°C and 5% CO₂. Then, 1 µM PGD₂ (Cayman Chemicals, Ann Arbor, MI, USA) was added or not to the cells for 20 h. At the end of the incubation, the cells were harvested and stained as described in the following section.

B cells from healthy individuals were cultured following isolation from fresh PBMCs (EasySep Human B Cell Isolation Kit) in a 37°C humidified incubator with 5% CO₂ while plated in a 96-well round-bottom plate (Sarstedt) in a concentration of 1.5 × 10⁵ cells per well. Cells were cultured in RPMI 1640 with GlutaMAX (catalog no. 61870036, Gibco) supplemented with 10% (v/v) heat-inactivated FBS (catalog no. 10270106, Gibco), penicillin-streptomycin (100 U/ml and 100 μg/ml, respectively; catalog no. 15140m, Gibco), sodium pyruvate (1 mM; catalog no. 11360070, Gibco), Hepes (10 mM; catalog no.15630106, Gibco), and 2-mercaptoethanol (0.05 mM; catalog no. 31350010, Gibco). In addition, all cultured B cells, irrespective of experiment, were supplemented with a “survival/mild proliferation” cocktail of IL-21 (50 ng/ml; catalog no. 200-21, PeproTech), CpG-B (2.5 μg/ml; catalog no. HC4039, Hycult Biotech), and sCD40L/CD154 (1 μg/ml; catalog no. 11343345, ImmunoTools). To mimic DDR in SLE B cells, IFN-α (catalog no. 11200-1, PBL Assay Science) was added to cells at 850 U/ml. For chemical ATR inhibition, berzosertib (i.e., ATRi; catalog no. S7102, Selleckchem) was added to cells at 2 or 5 μΜ, and for Chk1 inhibition, CHIR-124 (i.e., Chk1i; catalog no. HY-13263, Selleckchem) was added to cells at 50 nM (details on legends).

Chromosome preparations were obtained from short-term blood lymphocyte cultures following standard procedures described elsewhere [18 (link)]. Briefly, 1 mL of sodium heparin-stabilized peripheral blood was mixed with 9 mL culture medium containing RPMI-1640 with Glutamax (Gibco), 30% fetal bovine serum (R&D Systems Inc., Minneapolis, MN, USA), 1× antibiotic-antimycotic (Invitrogen, Waltham, MA, USA), and 1.4 µg/mL pokeweed mitogen (Sigma Aldrich, St. Louis, MO, USA). The cultures were grown for 72 h, harvested with demecolcine solution (final conc. 0.1 µg/mL; Sigma Aldrich), treated with optimal hypotonic solution (Rainbow Scientific, Windsor, CT, USA), and fixed in 3:1 methanol:acetic acid. Chromosome preparations were performed on clean wet slides, air dried and stored at −20 °C until needed.