Rpmi 1640

Mouse neuroblastoma Neuro2a (N2a) and human glioblastoma A172 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) and RPMI 1640 (Capricorn Scientific, Ebsdorfergrund, Germany) supplemented with 10% fetal bovine serum (FBS) and 1% antibiotics and incubated at 37 °C in the presence of 5% CO₂. Stable cells expressing shRNAs were established by transfection of shHuD plasmid (Santa Cruz Biotechnology, Inc., Dallas, TX, USA) or control plasmid under puromycin (Invitrogen™, Waltham, MA, USA) selection. Enhanced green fluorescent protein (EGFP) reporter was prepared by cloning the 3′UTR sequence of Ccl2 mRNA (533–806, 274 nt) into the pEGFP-C1 (BD Bioscience, Franklin Lakes, NJ, USA) vector. HuD overexpression plasmids (pHuD) were received as a gift from Prof. Alessandro Quattrone [18 (link)]. Transfection of small interfering RNAs (HuD siRNA (siHuD) and control siRNA (siCtrl)) (Genolution Pharmaceuticals, Inc., Seoul, South Korea) or plasmids were achieved using Lipofectamine™ 2000 (Invitrogen™) according to the manufacturer’s instructions.

Human breast cancer cell lines, MCF-7 and MDA-MB-468, were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). Cells were cultured in RPMI1640 (Capricorn scientific, Ebsdorfergrund, Germany) containing 10% fetal bovine serum (FBS, Hyclone, Logan, UT, USA), 100 U/mL penicillin and 100 μg/mL streptomycin (GenDEPOT, Barker, TX, USB) at 37 °C in a humidified incubator with 5% CO₂.

At the age of 30 weeks, mice were weighed, and anesthetized by inhalation of isoflurane. The body size was measured by measuring the length from nose-tip to tail-base before euthanasia by neck dislocation. The procedures were in accordance with the animal experiment regulations of the Philipps-University Marburg (Ex 17/2022). Bone marrow-derived cells (BMDC) were isolated postmortem from femurs of mice as described by Amend et al. (35 ), cultured for 24 h in RPMI-1640 medium, containing 1% penicillin and streptomycin (Capricorn Scientific GmbH) and 10% heat-inactivated fetal bovine serum (FBS) (Capricorn Scientific GmbH) and in an environment of 37°C and 5% CO₂. For BMDC differentiation to bone marrow-derivate MΦ (BMDM), BMDC in the supernatant were centrifuged (250 ×  g; 10 min) and then cultured in RPMI-1640 (Capricorn Scientific GmbH) supplemented with 10% heat-inactivated FBS (Capricorn Scientific GmbH), 1% penicillin and streptomycin (Capricorn Scientific GmbH), and recombinant mouse GM-CSF (20 ng/ml) (BioLegend, San Diego, CA) and allowed to grow for 6 days. Incubation of BMDM of mice with IFN-γ and LPS for 24 h lead to the differentiation into BMDM1-MΦ, and incubation of BMDM with IL-4 and IL-13 for 24 h lead to the differentiation into BMDM2-MΦ.

The investigated cell lines, PC-3 (human prostate adenocarcinoma) and HT-29 (human colorectal adenocarcinoma), were purchased from ATCC (Manassas, VA, USA). The cell culture medium RPMI 1640, the supplements FCS and L-glutamine, as well as PBS and trypsin/EDTA, were purchased from Capricorn Scientific (Ebsdorfergrund, Germany). Culture flasks, multi-well plates, and further cell culture plastics were from Greiner Bio-One (Frickenhausen, Germany) and TPP (Trasadingen, Switzerland), respectively. Resazurin used for the cell viability assays was purchased from Sigma-Aldrich (Taufkirchen, Germany).

The investigated cell lines, PC-3 (human prostate adenocarcinoma) and HT-29 (human colorectal adenocarcinoma) were purchased from ATCC (Manassas, VA, USA). The cell culture medium RPMI 1640, the supplements FCS and l-glutamine, as well as PBS and trypsin/EDTA were purchased from Capricorn Scientific GmbH (Ebsdorfergrund, Germany). Culture flasks, multi-well plates, and further cell culture plastics were purchased from Greiner Bio-One GmbH (Frickenhausen, Germany) and TPP (Trasadingen, Switzerland), respectively. Anti-proliferative and cytotoxic effects, respectively, of the compounds, were investigated by performing colorimetric MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and CV (crystal violet)-based cell viability assays (Sigma-Aldrich, Taufkirchen, Germany), respectively.

To analyze effects of FAP-IL-2v on the cellular cytotoxic activity of effector cells (ADCC) mediated by DB, a non-radioactive calcein-AM-based cytotoxicity assay was used, as previously described [10 (link)]. Briefly, leukocytes of healthy donors were cultivated for five days using RPMI 1640 (Capricorn Scientific GmbH, Ebsdorfergrund, Germany) supplemented with 2 mM stable glutamine, 0.3× P/S and 10% Sera Plus (PAN-Biotech GmbH, Aidenbach, Germany). To show the effects of FAP-IL-2v on ADCC, culture medium was supplemented with 1 µg/mL/day of FAP-IL-2v. Untreated leukocytes and leukocytes incubated with IL-2 (3000 IU/mL/day) served as controls. To induce ADCC against NB cells, DB (10 µg/mL) and an effector-to-target cell ratio of 40:1 were used. The GD₂-positive human NB cells LAN-1 (5000 cells/well) served as targets cells. The GD₂ specificity of ADCC was confirmed using the anti-idiotype Ab ganglidiomab [13 (link)]. The DB-independent cytotoxicity of leukocytes (AICC, antibody-independent cellular cytotoxicity) was evaluated by incubation of leukocytes with tumor cells with rituximab.

The mouse mammary epithelial HC11 cell line was purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA), and the mouse embryonic fibroblast NIH3T3 cell line was kindly provided by Dr. Young Bong Kim (Konkuk University, Seoul, Korea). HC11 cells were maintained in Roswell Park Memorial Institute 1640 medium (RPMI 1640; Capricorn Scientific, Ebsdorfergrund, Germany) supplemented with 10% fetal bovine serum (FBS; Merck, Kenilworth, NJ, USA) and 1% penicillin/streptomycin (Capricorn Scientific). NIH3T3 cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM; Capricorn Scientific) supplemented with 10% FBS and 1% penicillin/streptomycin. Both cell lines were grown in a humidified incubator at 37 °C under 5% CO₂ conditions.