Rpmi 1640 medium glutamax supplement

The human monocytic cell line THP-1 cells (TIB-202) were purchased from ATCC. Cells were cultured in 24 well plates (Corning) and differentiated into THP-1 derived macrophages 24 hours prior to infection with 40 ng/mL phorbol 12-myristate 13-acetate (PMA) in RPMI 1640 Medium GlutaMAX™ Supplement (Gibco) at 37°C and 5% CO2. Experiments were realized within 15 passages and cell viability was measured before experiments by trypan blue exclusion and was greater than 97%. CD14⁺ primary human monocytes were extracted from peripheral blood of 5 healthy donors anonymously provided by the French Blood Establishment (EFS, Lyon). CD14⁺ monocytes were purified from whole blood using an autoMACS^® Pro Separator, Whole Blood Column Kit and StraightFrom^® Whole Blood CD14 MicroBeads (Mitenyi) according to the manufacturer’s instructions. Cell viability was measured before proceeding to macrophage differentiation, by trypan blue exclusion and was always greater than 80%. CD14⁺ monocytes were then plated in 24 well plates (Corning) at a density of 400 000 cells per well and differentiated into macrophages for 5 days prior to infection with 50 µg/ml Rh-GMCSF (Miltenyi) in RPMI 1640 Medium GlutaMAX™ Supplement (Gibco) with 10% FBS (Sigma-Aldrich) at 37°C, 5% CO2.

An informed consent was given by the participants, according to the requirements of the institutional review board (Ethics Commission, Centre hospitalier universitaire Vaudois, CHUV). Cell line T1185B was derived from non-lymphoid metastasis of a melanoma patient at the Ludwig Institute for Cancer Research, Department of Oncology, University of Lausanne^{77 (link)}. Cell line was grown in RPMI 1640 Medium GlutaMAX™ Supplement (Gibco) with 0.55 mM L-arginine (Sigma), 0.24 mM L-asparagine (Sigma), 1.5 mM L-Glutamine (Gibco), 10 mM HEPES (Gibco), 10% of heat-inactivated fetal bovine serum (FBS), 100 U/mL penicillin and 100 μg/mL streptomycin (BioConcept). Snap-frozen tumor tissues from different regions of a lymph node of Mel-1 melanoma patient (clinical study: NCT03475134) were collected and stored at −80 °C. A cell line was generated from the same patient’s tumor at the CTE Biobank (CHUV) and grown in RPMI 1640 Medium GlutaMAX™ Supplement (Cat# 61870010, Gibco) with 10% of non-heat inactivated FBS, 100 U/mL penicillin and 100 μg/mL streptomycin. After in vitro expansion, both cell lines were trypsinized, washed twice in PBS and dry pellets containing 1 × 10⁸–2 × 10⁸ cells were collected and stored at −80 °C, before HLA-I immunoprecipitation (HLA-IP) workflow.