Cd45 microbeads kit

Lung cells were dissociated as previously described (Sharma et al., 2022 (link)). The right lung was dissociated to a single cell suspension with Multi Tissue Dissociation Kit 2 (130–110–203, Miltenyi) according to the manufacturer’s protocol using the gentle MACS Dissociator (Miltenyi). The cell suspension was filtered through a 70 μm strainer, pelleted by centrifugation, and cleared of red blood cells with ACK Red Blood Cell Lysis buffer (BP10-548E, Lonza). Live cells were enumerated using trypan blue exclusion counting on a Countess Automated Cell Counter Hemocytometer C10227 (Invitrogen). Lung cells were then used as described below for Fluorescence-Activated Cell Sorting (FACS) analysis. Additionally, ten million lung cells per animal were used for CD45⁺ enrichment using the CD45 MicroBeads kit (cat# 130–109–682, Miltenyi) per manufacturer’s instructions.

Surplus tumor tissue obtained at diagnostic biopsy or tumor resection was processed immediately after receipt in the histopathology laboratory (<1 hour after interventional radiology/surgical procedure). Tissue was minced using a scalpel and then incubated in RPMI 1640, supplemented with 10% fetal calf serum, 1% l-glutamine, and 1% penicillin/streptomycin, with collagenase IV (1.6 mg/ml; catalog no. 11410982; MP Biomedicals), for 30 min at 37°C, inverting the tube every 10 min. The digested tissue was passed through a 70-μm filter and incubated in 1× RBC lysis buffer (catalog no. 420301; BioLegend) for 10 min at room temperature. The obtained single-cell suspension was used for downstream processing. Part of the single-cell suspension was depleted of CD45⁺ cells to enrich for tumor cells using a CD45 MicroBeads kit (catalog no. 130-045-801; Miltenyi Biotec), following the manufacturer’s protocol. Both CD45 nondepleted and CD45-depleted single-cell suspensions were depleted of dead cells using a Dead Cell Removal kit (catalog no. 130-090-101; Miltenyi Biotec), following the manufacturer’s protocol. Obtained viable single-cell suspensions (CD45 depleted: two channels and CD45 nondepleted: one channel) were processed on the 10x Chromium platform.