Hts transwell 96 permeable support with 5.0 m pore polycarbonate membrane

Transwell plates (HTS Transwell^®-96 Permeable Support with 5.0 µm Pore Polycarbonate Membrane, Corning, Corning, NY, USA) were used. Supernatants of long-term treated NK cells were collected 24 h after medium exchange. Then, 235 µL of the supernatants was added to the lower chamber and 0.25 × 10⁶ of freshly isolated PBMCs and granulocytes in 80 µL were added onto the insert. After 5 h of incubation (37 °C, 5% CO₂), plates were incubated for 10 min at 4 °C, performed to induce detachment of monocytes. Cells were then removed from the lower chamber, transferred to a v-well plate, and pelleted by centrifugation. In the next step, cells were resuspended in 50 µL FACS-buffer containing an antibody mix of CD3 PerCP (1:100; Clone:SK7, BioLegend), CD56 BV421 (1:50; Clone:NCAM16.2; BD Biosciences), CD14 FITC (1:50 Clone: 61D3; Santa Cruz Biotechnology, Dallas, TX, USA), CD19 AF700 (1:50; Clone:HIB19; BioLegend), CD66b APC (1:200; Clone:G10F5; BioLegend) and CD11c PE (1:100; Clone: B-ly6; BD BioSciences) transferred to TruCount tubes (BD BioSciences) and incubated for 20 min at RT in the dark. Measurement of cells was performed using the BD LSRFortessa. Data were analyzed using FlowJo software (FlowJo) and absolute cell counts were calculated according to the manufacturer’s instructions.

Neutrophil chemotaxis was assessed using HTS Transwell-96 Permeable Support with 5.0 µm pore polycarbonate membrane (Corning). Per measurement, 50,000 neutrophils were placed in the upper chamber and incubated for 20 min at 37°C. The number of migrated neutrophils toward 5 nM fMLF (Glycotope, Germany) placed in the lower chamber was assessed using flow cytometry.