Rpmi 1640 media

Human Burkitt’s lymphoma (BL) cell lines DG-75 and RAJI and diffuse large B-cell lymphoma (DLBCL) cell lines SU-DHL-4 (GCB) and U-2946 (ABC) were purchased from the German Collection of Microorganisms and Cell Cultures (DSMZ, Braunschweig, Germany), cultured as recommended by the manufacturer in RPMI 1640 media (Biochrom, Berlin, Germany) supplemented with 10% heat-inactivated FCS (Biochrom, Berlin, Germany) and 100 µg/mL penicillin and streptomycin (Biochrom, Berlin, Germany). Cultured cells were regularly tested for mycoplasma contamination and authenticity (cell surface markers by flow cytometry).
AZD5153 (AZD), I-BET 151 (GSK1210151A) (I-BET) and Entospletinib (GS-9973) (Ento) were obtained from Selleck Chemicals (Absource Diagnostics GmbH, Munich, Germany) and prepared according to the manufacturer’s instructions to a 10 mM stock and stored at −80 °C until ready for use.
Cell lines (3.33 × 10 ⁵ cells/mL) were exposed to AZD, I-BET, Ento or DMSO (control) as mono substance (0.001 µM–10 µM) or in combination for up to 72 h (0.01 µM AZD, 0.1 µM I-BET, 1 µM Ento)

For the immunomodulatory analysis in vitro, femora, and humeri were harvested from C57BL/6 N mice. To isolate the bone marrow, the epiphyses were cut off from the bones and the bone marrow was flashed out into RPMI 1640 media (Biochrom, Berlin, Germany). The bone marrow was pushed through a 40 μm cell strainer to get a single cell suspension. Residing erythrocytes were lysed for 4 min at room temperature (RT) in ACK lysing buffer (Gibco Life Technologies GmbH, Darmstadt, Germany). After centrifugation, cells were resuspended in 10 ml RPMI 1640 media and counted.

For standard proliferation assays 2 × 10⁵ PBMCs were cultured in R10 medium (composed of RPMI 1640 media (Biochrom, Berlin, Germany) supplemented with 10% FCS, 1 mM l-glutamine, 50 U/mL penicillin, 0.5 mM sodium pyruvate, and 50 μg/mL streptomycin) for 48 h with PHA, ConA, PWM, and immobilized CD3 mAb (iCD3) either with or without soluble co-stimulatory anti-CD28 (1 μg/mL), respectively. For immobilization 100 µL anti-CD3 (OKT3, 2.5 μg/mL; purified from hybridoma supernatant) was adsorbed onto Nunc Maxi-Sorb 96-well plates (Sigma Aldrich, St. Louis, MO, USA) for 60 min at 37 °C. The plate was washed three times. Proliferation of CD4⁺ T cells was assessed after magnetic enrichment of the target cells from frozen PBMC after thawing using the IMag CD4 T Lymphocyte Enrichment Set (Becton Dickinson, Heidelberg, Germany) and incubating 5 × 10⁴ cells/well with media, PHA and iCD3 either with or without soluble co-stimulatory anti-CD28 (1μg/mL), respectively, in triplicates for 72 h. Radioactive thymidine was added to each well. 24 h later cells were harvested and incorporated radioactivity was determined using a beta-counter.