Macsquant analyser 10 flow cytometer

Binding of biotinylated scFv(AM1)-P-BAP to PSCA-positive target cells and isogenic parental cells was analyzed by flow cytometry. 2 × 10⁵ cells were stained with 5 μg scFv-P-BAP for 1 h at 4 °C, followed by secondary anti-biotin-VioBlue antibody (Miltenyi Biotec, Cologne, Germany). Cells stained with the secondary antibody alone served as the control. To evaluate the targeted delivery of DNA, 2 × 10⁵ 293T^PSCA cells were incubated with scFv-P-BAP hybrid polyplexes loaded with Cy3-labelled plasmid DNA (Mirus Bio, Madison, WI, USA, plasmid # MIRUMIR7904, 2.7 kb) for 4 h at 37 °C. Cy3-plasmid-loaded hybrid polyplexes conjugated with control scFv(MR1.1)-P-BAP were included as a negative control. Subsequently, the cells were washed with 0.1% Heparin/PBS (Sigma-Aldrich). To evaluate the transfection efficiency, 1 × 10⁵ 293T^PSCA were incubated with PPI:MC-GFP dendriplexes containing 1 µg MC-DNA and PPI at different mass ratios for 48 h. At least 2 × 10⁴ cells were measured by MACSQuant Analyser 10 flow cytometer (Miltenyi Biotec) and analyzed by FlowJo software version 10.1 (TreeStar, Ashland, OR, USA).

Human peripheral blood mononuclear cells were isolated from buffy coats using density gradient. For this, 25 ml of blood from healthy donors (German Red Cross, Frankfurt, Germany) were mixed with the same amount of Hank’s balanced salt solution (Thermo Fisher Scientific, Oberhausen, Germany) and were layered over 15 ml of Biocoll (Merck, Darmstadt, Germany) in Sep-Mate™-50 tubes (Stemcell Technologies, Cologne, Germany). Tubes were centrifuged (1200 g, 10 min, RT), and human peripheral blood mononuclear cells were isolated from the interphase, washed with 2 mM EDTA/PBS four times and counted using a MACSQuant® Analyser 10 flow cytometer (Miltenyi Biotec, Bergisch Gladbach, Germany). Defined amounts of cells were dissolved in 0.5% bovine serum albumin/2 mM EDTA/PBS and incubated with 25% (v/v) human CD14 MicroBeads for 15 min at 4 °C. Afterwards magnetically labelled cells were separated via magnetic cell separation with LS columns (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer’s protocol. Cell count was determined with flow cytometry.