Accutase solution

The tests were carried out with murine melanoma B16-F10 (given by Prof. J. Dulak, Department of Medical Biotechnology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Cracow, Poland; authenticated in 2021, ATCC) and renal carcinoma RenCa (CRL-2947™, ATCC, Manassas, VA, USA) cell lines. Cells were maintained in standard culture conditions: humidified atmosphere containing 5% CO₂ at 37 °C, using RPMI medium—RPMI-1640 GlutaMaxTM (ThermoFisher Scientific, Waltham, MA, USA), with 10% FBS (v/v) (ThermoFisher Scientific). Cells were passaged using Accutase solution (Biolegend, San Diego, CA, USA) after confluence reached 70–80%. Cells used were Mycoplasma free as assayed with PCR Mycoplasma Test (Biomedica, Piaseczno, Mazowieckie, Poland) and did not exceed 15th passage.

THP1-Dual™ WT, THP1-Dual™ KO-STING, THP1-Dual™ KO-cGAS, THP1-Dual™ KO-TBK1 cell lines were cultured in 10% (v/v) FBS supplemented RPMI 1640 medium containing 100 μg/ml zeocin and 10 μg/ml blasticidin (In vivogen, France). For macrophage differentiation of THP-1 Dual cells, PMA was used at high concentration (50ng/ml) overnight or low concentration (5ng/ml) for 48 hours. After PMA treatment, to equalize the initial macrophage number for all cell lines, cells were detached by using accutase solution (Biolegend, U.S.A.), counted on Novocyte 2060R flow cytometer (ACEA Biosciences, U.S.A.) and distributed to 96-well plates at a density of 1x10⁵ cells/well in Leishmania Infection Medium (RPMI 1640 supplemented with 2% FBS, 20mM HEPES, 100 units/ml penicillin, 100µg/ml streptomycin). Multiplicity of infection (MOI) was optimized as 1:10 (THP-1:stationary phase L. major). After 6h of infection, wells were washed with DPBS to remove excess parasites and fresh 10% (v/v) FBS supplemented RPMI 1640 medium was added on to the infected THP-1 cells. Percent infection rates and parasite loads (MFI) were based on the eGFP signal and were quantitated on a Novocyte 2060R flow cytometer (ACEA Biosciences, U.S.A.). In certain experiments, Amlexanox (In vivogen, France) was added into the THP-1 growth medium 1 hour prior to infection at indicated concentrations or 24 hours post-infection.

Apoptosis was performed per prior methods (2 (link)). Briefly, MDMs were plated at a density of 1 × 10⁶/ml in 12 well plates. Apoptosis was measured by flow cytometry and fluorescence-activated cell sorting (FACS) analysis using APC Annexin V (Biolegend, 640920) and propidium iodide (BioLegend 421301). MDMs were detached by Accutase solution, collected, washed, re-suspended in 100 μl of Annexin V Binding Buffer (Biolegend 422201), and then stained with 5 μl of Annexin V and 10 μl propidium iodide for 15 min at room temperature in the dark. The percentage of viable and apoptotic cells was calculate using flow cytometry (BD LSR II Flow Cytometer).