Rpmi 1640 glutamax medium

iNKT cells from human DLIs were expanded in iNKT-cell culture medium consisting of RPMI 1640 GlutaMAX™ Medium (ThermoFisher Scientific, Waltham, Massachusetts, USA), 10% FBS (fetal bovine serum, Biochrom, Berlin, Germany), 100 IU/ml penicillin-streptomycin (Lonza, Basel, Switzerland), 5.5 μM 2-mercaptoethanol (Roth, Karlsruhe, Germany), 0.1 mM non-essential amino acids (NEAA, Gibco, New York, New York, USA), 10 mM HEPES (Gibco) and 1 mM sodium pyruvate (Gibco). Donor lymphocytes were co-incubated with 100 ng/ml α-GalCer (Sigma-Aldrich, St. Louis, Missouri, USA) and 100 IU/ml recombinant human interleukin 2 (rhIL-2, Novartis, Basel, Switzerland). At day 7, rhIL-2 (100 IU/ml) and α-GalCer (100 ng/ml) was added to the culture and iNKT cells were re-stimulated with irradiated (30 Gy, cesium-137 irradiator Gammacell 1000, Atomic Energy of Canada Limited, Chalk River, Ontario, Canada) and glycolipid-pulsed autologous or allogeneic peripheral blood mononuclear cells (PBMCs) for another 7 days. Thereafter, iNKT-cell expansion was completed. At days 7 and 14, viability and percentage of DLI-iNKTs were measured by flow cytometry.

Luciferase-expressing cell lines derived from human gastric adenocarcinoma (MKN-45), breast adenocarcinoma (MCF-7) and hepatocellular carcinoma (HuH-7), were obtained from the Japanese Collection of Research Bioresources (Osaka, Japan). MKN-45 cells were maintained in RPMI1640 Glutamax medium (Thermo Fisher Scientific, Tokyo, Japan) supplemented with 10% fetal bovine serum (FBS; Equitech-Bio, Kerrville, TX) in an atmosphere of 5% CO₂ at 37 °C. MCF-7 and HuH-7 cells were maintained in Dulbecco’s minimal essential medium (DMEM; Thermo Fisher Scientific) instead of RPMI1640. Two Chinese hamster ovary (CHO) cell lines, human CEA-expressing CHO-CEA and human EpCAM-expressing CHO-EpCAM cells, have been described previously [5 (link)], and were cultured in α-modified minimum essential medium (α-MEM; Thermo Fisher Scientific) supplemented with 10% FBS and 2 mM glutamine. Murine tumor endothelial cell line 2H-11 was obtained from the American Type Culture Collection (Manassas, VA) and was maintained in DMEM supplemented with 10% FBS and 2 mM glutamine (Wako Pure Chemicals, Osaka, Japan). Primary human breast CAF derived from an infiltrating ductal-carcinoma tissue was purchased from Asterand (Detroit, MI) and maintained in DMEM supplemented with 10% FBS and penicillin–streptomycin.

Three basal-like TNBC cell lines, MDA468, HCC1143, and HCC1937, were employed to validate the results of PDX studies. MDA468 cells were provided by Dr. Youngman Oh (VCU Department of Pathology); HCC1143 and HCC1937 cells were obtained from the American Type Culture Collection (ATCC). Cell lines were cultured in RPMI-1640 GlutaMAX medium (ThermoFisher Scientific) supplemented with 10% FBS and penicillin/streptomycin.

Kidney cancer cell lines with different PTEN status were purchased from ATCC (Manassas, VA, USA). PTEN wild-type: murine Renca (Cat# CRL-2947, ATCC, USA), human Caki-1 (Cat# HTB-48, ATCC, Manassas, VA, USA) and PTEN mutant 786-O cell line (Cat# CRL-1932, ATCC, Manassas, VA, USA). All cancer cells were cultured in RPMI-1640 GlutaMax™ medium (Thermo Fisher Scientific, Waltham, MA, USA), with 10% Fetal Bovine Serum (FBS) (Thermo Fisher Scientific, Waltham, MA, USA). Murine brain derived mature endothelial cells (ECs) (MBr MEC FVB) [34 (link)], were cultured in OPTI MEM medium (Thermo Fisher Scientific, USA), with 2% FBS (Thermo Fisher Scientific, Waltham, MA, USA). All cell lines and were passaged at 80% confluence by detaching with Accutase solution (Biolegend, San Diego, CA, USA). Cells used in the experiments were Mycoplasma free as assayed with PCR Mycoplasma Test (Biomedica, Piaseczno, Poland) and did not exceed the 15th passage.

Single cell suspensions were obtained by dispersion of murine spleens through a 100 μm cell strainer (BD Biosciences), followed by erythrocytes lysis. B cells were purified from splenocytes using anti-CD19 microbeads (Miltenyi Biotec) following the manufacturer’s protocol. 3x10⁵ CD19+ B cells were cultured in 96-well round bottom plates (Sigma-Aldrich) in 200 μL RPMI 1640 Glutamax medium (Thermo Fisher Scientific) containing 5% heat-inactivated Fetal Calf Serum (FCS, Bodinco), 50 μM β-2-mercapthoethanol (Sigma-Aldrich), 100 U/mL penicillin and 100 μg/mL streptomycin (Sigma-Aldrich). Cells were stimulated with SEA (20 μg/mL), SEA fractions (25 μL), or recombinant molecules (SmTrx1, Sm14 or SmVAL28, 1–50 μg/mL) for a total of 48 hours. After 44 hours, supernatants were collected and kept at -20°C for later cytokine analysis by ELISA. Cells were restimulated during the last 4 hours with 100 ng/mL phorbol 12-myristate 13-acetate (PMA), 1 μg/mL ionomycin and 10 μg/mL Brefeldin A (Sigma-Aldrich) before flow cytometric analysis.

Cell culture was performed using aseptic technique at 37°C in a humidified atmosphere in 5% CO₂, unless otherwise specified. The human ovarian carcinoma cell line IGROV1 naturally over-expressing folate receptor alpha (FRα) was grown in RPMI 1640 GlutaMAX™ medium (Thermo Scientific) supplemented with 10% fetal calf serum (FCS). The human breast cancer cell line MDA-MB-231 was grown in DMEM GlutaMAX™ medium (Thermo Scientific) supplemented with 10% FCS. The permanently transfected murine myeloma cell lines SP2/0-MOv18 specific for FRα and SP2/0-SF25, recognizing a colon carcinoma antigen (23 (link)), were cultured in Dulbecco’s Modified Eagle’s Medium plus 10% FCS as previously described (24 (link)). The human embryonic kidney cell lines, Expi293F cells, were cultured in serum-free Expi293 expression medium (Thermo Scientific) on a Stuart orbital shaker at 125 rpm at 8% CO₂.

Bone marrow-derived macrophage (BMDM) progenitors were obtained by flushing mouse femurs and tibias, followed by erythrocyte lysis with red blood cell lysis buffer (#B00003, Roche). BMDMs were obtained by a 7-day differentiation of progenitors in RPMI 1640 GlutaMAX™ medium (#61870-010, ThermoFisher Scientific) supplemented with 10% heat-inactivated fetal calf serum (#A15-102, PAA) and 10% L929-conditioned medium as a source of M-CSF [hereafter called complete medium (CM)]. THP-1 human monocytes (ATCC, TIB-202) were cultured in RPMI supplemented with 10% heat-inactivated fetal calf serum, penicillin, and streptomycin (#15140122, ThermoFisher Scientific). They were differentiated into macrophages by addition of 2 ng/ml phorbol 12-myristate 13-acetate (#P8139, Sigma) for 3 days.