Macs ms column

Positive selection of biotin⁺ PMCs was performed using microbeads. Cells were magnetically labeled with streptavidin microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany, 130‐048‐102), and passed through MACS MS columns (Miltenyi Biotec, 130‐042‐201), which were placed in the magnetic field according to the manufacturer's instruction. Biotin⁺ PMCs were flushed out from the column and passed over a new column to increase the purity. To remove contaminated MΦs, the samples were further passed through CD11b microbeads column (Miltenyi Biotec, 130‐049‐601) and anti‐F4/80 microbeads column (Miltenyi Biotec, 130‐110‐443).

An optimized pipeline for viable cell recovery and more balanced cell-type representation was used to process lymph nodes for scRNA-seq. Lymph nodes were enzymatically digested using a protocol that maximizes the recovery of myeloid and stromal cells while maintaining high viability⁴⁰ . In brief, lymph nodes were placed in RPMI with collagenase IV, dispase and Dnase I at 37 °C, and cells were collected once they were detached. The cells were then immediately placed on ice and washed with PBS supplemented with 2 mM EDTA and 0.5% biotin-free BSA, then filtered through a 70 µm cell strainer. Cells were incubated with Fc blocking antibodies 4 °C, then with a biotinylated anti-CD3 and anti-CD19 antibody cocktail. Antibodies were used at a dilution of 1:100. Streptavidin microbeads were then added and the cells were magnetically sorted using MACS MS columns according to the manufacturer’s protocol (Miltenyi Biotec). After cell sorting, a small fraction of the CD3⁺ or CD19⁺ cells was pooled with CD3^–CD19^– cells for more balanced representation of all cell types and proceeded immediately to scRNA-seq.

CD14-positive cells were isolated from freshly isolated PBMC using CD14 microbeads and MACS MS columns according to the manufacturer’s instructions (Miltenyi Biotec, Bergisch Gladbach, Germany). Isolated CD14-positive cells were cultured in RPMI-1640 with 10% FBS.

The hemolyzed SVF was applied to a magnetic-activated cell sorting (MACS) system using CD45 and CD31 microbeads (Miltenyi Biotec; #130-045-801 and #130-091-935) consecutively, according to the manufacturer’s instructions. The separation buffer (MACS buffer) consisted of 0.5% (w/v) BSA (Sigma-Aldrich; #A8806) and 2 mM EDTA in HBSS. Both the negative and positive fractions separated using MACS MS columns (Miltenyi Biotec) were collected. CD45⁻CD31⁺ (AEPC-enriched fraction) and CD45⁻CD31⁻ (ASC-enriched fraction) fractions were seeded on cell culture plates (Corning) at 1 × 10⁴ cells/cm² in cell culture medium (EGM-2MV BulletKit; Lonza; #CC-3202) (Supplemental Table S1) at 37 °C in 5% CO₂ and 95% air. A second MACS procedure using only CD31 microbeads was later applied to further purify AEPCs.

Peripheral blood mononuclear cells (PBMCs) were isolated by centrifugation (500 × g, 30 min, RT) in a Histopaque-1077 density gradient (Sigma–Aldrich) of buffy coats obtained from healthy donors from Regional Centre of Blood Donation and Blood Treatment in Lodz in accordance with applicable law. Peripheral blood monocytes (PBMs) were purified from the PBMC fraction by magnetic separation on MACS MS columns (Miltenyi Biotec, Germany) using MACS CD14 MicroBeads according to the manufacturer’s protocols. PBMs were maintained in RPMI 1640 medium (with ATCC modification) supplemented with 10% human AB serum (Sigma–Aldrich) and pen/strep for use in subsequent procedures.