Human trustain fc block

Isolated neutrophils were transferred to FACS buffer (2% FBS in 1X PBS) at 1 × 10⁶ cells in 200 μL. For tumor studies, bone marrow was flushed from tumor-bearing and saline-injected tibiae using a syringe and excess cells were further flushed out of the marrow with EasySep buffer. Cells were treated for 2–3 min in 1X Red Blood Cell Lysis Buffer (BioLegend) at room temperature, washed in 1X PBS, and counted for antibody staining. For staining, cells were incubated on ice with 1 μL per 10⁶ cells in mouse or human TruStain Fc block (Biolegend) for 10 min. Fluorophore-conjugated antibodies were added at a maximum of 1 μL per 10⁶ cells (Human-APC/Cy7-CD11b, PE/Cy7-CD14, FITC-CD15, PerCp-Cy5.5-CD10; Mouse- APC-CD45, FITC-CD11b, PE-Ly6G, PerCp-Cy5.5-Ly6C). Cell viability dye, Live/Dead (Invitrogen), was added at a concentration of 0.2 μL per 10⁶ cells. Stained cells were incubated with antibody on ice in the dark for 20 min and rinsed with 1X PBS. Cells were fixed by incubation in 1% formaldehyde in 1XPBS for 30 min in the dark and rinsed with 1X PBS. Prior to analysis, cells were reconstituted in FACS buffer. For all analyses, single cells were gated and marker expression analyzed in myeloid cells.

Peripheral blood samples were collected with Li-Heparin Monovettes (Sarstedt) and processed within 3 h. Red blood cells were eliminated by osmotic lysis with RBC lysis buffer as described before [2 ]. Leucocytes were then washed, re-suspended in FACS buffer (DPBS + 10% FCS). Unspecific binding of antibodies to cell surface receptors was blocked with Fc Block (1:20, BioLegend) or human TruStain Fc Block (BioLegend) for 20 min at 4 °C. Leucocytes were stained with antibodies specific for CD3 (1:50, BioLegend; UCHT1-PB), CD4 (1:100, BioLegend RPA-T4-PE), CD8 (1:100, BioLegend, SK1-APC), and CD69 (1:50, BioLegend; FN50-FITC) for 25 min at 4 °C in the dark. After washing, the stained cells were fixed for 20 min in 2% PFA, filtered through a 70 µM cell-strainer and characterized by flow cytometry on a LSRII flow cytometer (BD) using a combination of unstained cells, internal negative population controls and single-color staining for color compensation. Therefore, isotype-matched, host-matched monoclonal antibodies were used as negative control: mouse IgG2a, κ (BioLegend; MOPC-173-PE-Cy7), mouse IgG1, κ (BioLegend; MPOK-21-PB), mouse IgG1, κ (BioLegend; MOPC-21-PE), mouse IgG2a, κ (eBioscience; P3.6.2.81-APC). Flow cytometry data was processed with FACS Diva™ software (BD).

Tissues were homogenized and cells were released by using the gentleMACS™ Octo Dissociator with Heaters and the human tumor dissociation kit (both from Miltenyi Biotec), according to the manufacturer protocols. Afterwards, the cells were washed two times with F12-DMEM medium (Gibco) and collected by centrifugation at 300g for 5 minutes. The single-cell suspensions were further generated by passing the resuspended cells through the 70 mm cell strainers. The single cell suspensions were then further stained with the Zombie Aqua Fixable viability dye (1:100, Biolegend, 423101) at room temperature for 20 min, then washed with phosphate-buffered saline (PBS). The cells were incubated with Human TruStain Fc block (1:100, Biolegend, 422302) for 10 min to limit unspecific antibody binding, then stained for 20 min with anti-EPCAM (1:40, Biolegend, 324206) and anti-CD45 (1:40, Biolegend, 304021) in FACS buffer (PBS + 0.5% Bovine Serum Albumin). The cells were subsequently washed and resuspended in FACS buffer. Fluorescence-activated cell sorting (FACS) using an influx flow cytometer (BD Biosciences) was performed to sort live EPCAM+CD45- single-cells into 384 well plates for Smart-Seq3 analysis. The list of antibodies is provided in Supplementary Data 2.

BiKE and scFv-Fc binding to NK cells or tumor cells was analyzed by flow cytometry. Proteins bound to tumor cells via the anti-TAA scFv were detected with antibodies targeting the C-terminal His₆ tag. Antibodies targeting the N-terminal HA tag were applied to assess binding to NK cells via the anti-CD16A (BiKE) or IgG1Fc (scFv-Fc) moiety.
1 × 10⁵ cells were incubated with Zombie Violet fixable viability dye (1:5000, BioLegend) for 15 min in the dark. For NK cells, Human TruStain Fc block (1:40, Biolegend) was added. Cells were washed and incubated with purified vBiKEs (2 µL) or MV-BiKE and MV-scFv-Fc virus suspensions (20 µL) for 30 min on ice in the dark. NK cells were stained with anti-CD16-BV650 (clone 3G8, 1:100, BioLegend) in parallel. After washing, cells were labeled with anti-HA FITC (clone GG8-1F3.3.1, 1:20, Miltenyi Biotec) or anti-His₆ FITC (clone 13/45/31, 1:20, Dianova). Samples were fixed with 1% PFA in DPBS and analyzed by flow cytometry. Unstained and single stained controls were used to control for unspecific antibody binding.