Pre separation filter

Fresh PBMCs from two healthy donors were separated from whole blood samples using Ficoll-Paque^TM. CD14+ monocytes were selected using MACS human CD14 MicroBeads (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany), pre-separation filters (Miltenyi), and LS columns (Miltenyi) following the manufacturer’s recommendations. To induce M0 macrophages, isolated CD14+ monocytes were seeded onto FBS-coated 24-well plates at a density of 1 × 10⁵ cells/cm². Seeded cells were cultured for 7 days in RPMI 1640 media supplemented with 20% FBS and 100 ng/ml M-CSF (BioLegend, San Diego, CA, USA). On day 7, M-CSF-containing medium was removed, and appropriate stimulating media containing 100 ng/ml LPS (Sigma-Aldrich, St. Louis, MO, USA) and 20 ng/ml IFN-γ (BioLegend) for M1 induction or 20 ng/ml IL-4 (BioLegend) and 20 ng/ml IL-10 (BioLegend) for M2 induction were supplied. After an additional 48 h of culture, cells were collected by gentle scraping and then taken for fluorescence-activated cell sorting (FACS) analysis or scRNA-seq. The morphological changes that occurred during differentiation were observed every 2 days under a microscope. A fraction of the cells was triple-stained with PE/Cy7 anti-human CD14 antibody (BioLegend), FITC anti-human CD80 antibody (BioLegend), and PE anti-human CD163 antibody (BioLegend) and analyzed by FACSVerse and FACSuite v1.2 (BD Biosciences).

For crosslinking of the experimental samples, the harvested and processed tumor cell suspensions were subjected to a fixation protocol using DSP (Thermo Fisher Scientific, 22585). DSP de-crosslinking was achieved using DTT (Sigma Aldrich, Buchs, Switzerland, D9779), as described in detail by Attar et al. [7 (link)]. Briefly, a DSP stock solution (50×) was prepared in 100% anhydrous DMSO (50 mg/mL, stored at −80 °C). Immediately before use, the 50× DSP stock solution was diluted with PBS to working concentration (1 mg/mL) by adding 490 μL PBS to 10 μL DSP (50× stock) dropwise using a 200 μL pipette while vortexing, ensuring minimal precipitation. In the case of substantial precipitation, the dilution was started over with a new DSP stock aliquot. The 1× DSP solution was filtered using a 30 μm filter (Pre-Separation Filters, Miltenyi Biotec) and placed on ice. A total of 200,000 cells were dispensed into a 1.5 mL Eppendorf tube. The cells were then pelleted and washed twice with PBS. The cell pellet was gently resuspended in 200 µL 1× DSP and incubated at 20–22 °C for 30 min. To quench the crosslinker, 4.1 µL of 1 M Tris HCl, pH 7.5 (final concentration 20 mM) was added and gently mixed by pipetting. The fixed cells were stored at 4 °C until further processing. For de-crosslinking, DTT was added to a final concentration of 50 mM and incubated at 37 °C for 30 min.

A dissociated single-cell suspension was passed through a 30-µm nylon mesh (Pre-Separation Filters; Miltenyi Biotec #130-041-407) to remove cell clumps that could clog the column. After counting cell numbers, an appropriate number of cells was pipetted, which was defined as pre-separation interstitial cells. The remaining cells were pelleted and resuspended in 80 µl of MACS buffer (PBS pH 7.2, 0.5% BSA and 2 mM EDTA) and 20 µl anti-CD45 MicroBeads (Miltenyi Biotec #130-052-301) or 20 µl anti-F4/80 MicroBeads (Miltenyi Biotec #130-110-443) per 10⁷ total cells. The mixture was incubated in the dark at 4 °C for 15 min, washed, and resuspended in MACS buffer. Cell suspension was applied onto the pre-balanced LS column (Miltenyi Biotec #130–097-679) that was placed in the magnetic Separator. Then the LS column was removed from the separator and the magnetically labeled cells were immediately flushed out, which were defined as the CD45⁺ or F4/80⁺ fraction. After incubation with anti-CD45 MicroBeads, unlabeled cells which passed through the LS column and LD column (Miltenyi Biotec #130-042-901) were defined as the CD45^- fraction. Each fraction was seeded in 24-well plates with or without glass coverslips and maintained in a humidified atmosphere (5% CO₂, 95% air) at 37 °C for 3 days or 6 days in culture media (10% FBS in RPMI-1640).