70 μm filter

72 h after electroporation, cells were trypsinized, counted, and resuspended at the concentration of 3-5 × 10⁶ cells/ml in an appropriate volume of sorting buffer (phosphate-buffered saline (PBS)/0.5% bovine serum albumin (BSA)/2 mM EDTA and 100 units/ml penicillin/streptomycin (Life Technologies)) for flow cytometry analysis. Immediately before cell sorting, samples were filtered through a 70-μm filter (Miltenyi Biotec) to remove any clumps or aggregates. Sorting was performed in sterile conditions throughout the experiments in the sorting buffer described above recovering at least 0.5 × 10⁶ cells per experimental condition. Cell sorting was carried out in a Synergy 2 L instrument (Sony Biotechnology Inc.); flow cytometry was performed in a BD LSR Fortessa analyzer (BD Biosciences) and FACSDiva software was used. Proper electronic gates of side scatter and forward scatter parameters were set in order to exclude cell debris and dead cells. The SK-N-MC or HEK293 GFP negative control cells were analysed in order to assess the minimal fluorescein isothiocyanate baseline. Sorting was performed in sterile conditions throughout the experiment. Sorted cells were seeded individually per well in a 96 well-plate containing DMEM supplemented medium.

The excised primary tumor and spleen from each mouse were minced and digested enzymatically using the MACS Tissue Dissociation Kits (Miltenyi Biotec, Auburn, CA), according to the manufacturer's guidelines. After dissociation, each sample was filtered through a 70 μM filter (Miltenyi Biotec) and centrifuged at 300 ×g for 10 min; the cell pellet was resuspended in RPMI-1640. Each single-cell suspension was split into two. One half of the tumor single cell suspension was treated with Red Blood Cell Lysis Solution (Miltenyi Biotec), washed twice, and counted.
Spleen single-cell suspensions and the other half of the tumor single cell suspensions were layered over Ficoll-Paque premium (GE Healthcare Life Sciences, Pittsburgh, PA) and centrifuged at 400 ×g for 40 min at 20°C to allow density gradient separation of the leukocytes. After Ficoll, the leukocytes (buffy coat) were harvested, washed twice with PBS, and counted.
Single-cell suspensions were stained following standard protocols. Briefly, cells were washed using FACs buffer (PBS, 2% FBS, and 0.02% sodium azide). Cell pellets were resuspended, blocked using Rat IgG and Mouse IgG (Jackson ImmunoResearch, West Grove, PA), washed, and incubated with the appropriate antibodies described below. After incubation, the cells were washed, fixed in paraformaldehyde, and stored at 4°C.

To isolate whole bone marrow cells, long bones of 8–10-week-old C57BL/6J mice were triturated with mortar and pestle in PBS supplemented with 0.5% bovine serum albumin and 2 mM EDTA. Cells were filtered through a 70-μm filter (Miltenyi Biotech), washed, and centrifuged (10 min; 300 g; 4 °C). After erythrocyte lysis in hypotonic NH₄Cl − buffer, cells were filtered through a 30-µm filter (Miltenyi Biotech). For neutrophil isolation the mouse Neutrophil Isolation Kit (130-097-658, Miltenyi Biotec) was used according to the manufacturer’s instructions. Bone marrow derived non-neutrophils that were retained in the separator column after neutrophil isolation were plated in non-tissue culture treated dishes and treated with 20 ng/µl murine M-CSF (315-02, Peprotech) for 7 days to generate macrophages^{56 (link)}.

PC were enriched from BM mononuclear cells, using a magnetic cell-sorting CD138 MicroBeads kit (Miltenyi Biotec; BergischGlabach, Germany) according to the manufacturer’s protocol. BM samples were filtered with a 70 μm filter (Miltenyi Biotec), and suspended in RPMI solution (Dutscher; Brumath, France). After centrifugation (160 G), and removal of the supernatant, mononuclear cells were marked with CD138 microbeads at 4 °C for 15 min. The cells were then separated on a column kit separator (Miltenyi Biotec). PC enrichment was controlled on a cytospin slide by a cytologist. The median efficiency of CD138 selection was 97.5%.

Peripheral blood was collected in Cell Preparation Tube (CPT) vacutainers with sodium citrate (Becton Dickinson) and centrifuged according to the manufacturer’s recommendations for separating mononuclear cells from whole blood. PBMCs were washed with Dulbecco’s phosphate buffered saline (PBS) and frozen in RPMI 1640 media with 10% fetal bovine serum and 10% dimethyl sulfoxide. Frozen, isolated PBMCs were quickly thawed in a 37 °C water bath. The thawed PBMCs were diluted into 10 mL of flow buffer (PBS, 5% FBS, 2 mM EDTA, 0.09% sodium azide). The suspension was passed through a 70 μM filter (Miltenyi Biotech, Auburn, CA, United States) into a fresh tube. The cellularity of the filtered suspension was counted on a ThermoFisher Countess (ThermoFisher Scientific, Waltham, MA, USA). Cells were pelleted (800× g for 7 min) and the supernatant aspirated. The cells were resuspended in a flow buffer at 1 × 10⁷ cells/mL. 3 × 10⁶ cells in a total of 0.3 mL were set aside for PrimeFlow^TM processing. The remaining cells were washed two times with PBS then lysed in 50 μL RIPA buffer with cOmplete and PhosSTOP added (Millipore-Sigma, St. Louis, MO, USA). Lysates were clarified by centrifugation at 12,000× g for 12 min at 4 °C, then the clarified lysates were stored at −80 °C.