4 6 diamidino 2 phenylindole dapi

For immune fluorescence imaging, cells were seeded and differentiated in 8-well chamber slides (Falcon) and infected with fluorescent (Ypet) bacteria. Afterwards, cells in chamber slides were washed with PBS and fixed with 4% paraformaldehyde for 10 min. Cells were then permeabilized with 0.5% saponin (Sigma-Aldrich) for 30 min, stained with Alexa-flour-647-labeled CD71 (TFR1) antibody (Biolegend) for 2 h, and 4′,6-diamidino-2-phenylindole (DAPI, BioLegend) for 30 min. After staining, samples were mounted with Faramount Mounting Medium (Dako). Fluorescence microscopy was performed immediately after sample preparation using a VS120-S6 fluorescence microscope (Olympus). Images were captured with a 40-x objective using 387/440 nm (DAPI), 485/525 nm (Ypet), and 650/684 nm (Alexa-flour-647) lasers and filters, under identical exposure times for every sample. Fluorescence intensity of Alexa-flour-647 was determined with the CellSense (Olympus) program for at least 100 cells per sample.

The production of polyclonal antibody against rfBD was performed as previously indicated [31 (link)]. The BALB/c mice were immunized four times at biweekly intervals with 20 mg purified rfBD protein. Following four boosts, the serum samples were obtained and anti-rfBD polyclonal antibodies were purified using protein G agarose affinity chromatography (Thermo). The sterile PBS was injected into the abdominal cavity of the flounder and then withdrawn with a syringe. After being washed with PBS, 20 μL of peritoneal cells (5 × 10⁶ cells mL⁻¹) was dropped and settled on APES-coated slides for 1 h. The cells were fixed by 4% formaldehyde (Thermo) for half an hour. After incubation with 5% BSA for 2 h at room temperature, the peritoneal cells were treated with anti-rfBD for 1 h. After washing three times with PBS, the peritoneal cells were treated with Alexa Fluor 488–conjugated goat anti-mice IgG (1:1000, Sigma, Nawa Prefecture, Japan) for 45 min at 37 °C in the dark. After washing three times with PBS, the peritoneal cells were counter stained with 4,6-diamidino-2-phenylindole (DAPI, Bio-Legend, Santiago, Chile) and then viewed using a Zeiss fluorescence microscope (Zeiss, Jena, Germany).

Dissociated retinal cells from AAV8(Y733F).CAG.GFP.WPRE-injected mice were stained with the viability dye ViaKrome 405 (Beckman Coulter) at 1/1,000 dilution in PBS for 20 min on ice. Cells were subsequently incubated with TruStain FcX PLUS Fc block (2.5 μg/mL) (BioLegend, London, UK) on ice for 10 min. Cells were washed with staining buffer prior to cell surface antigen staining. Retinal cells from AAV8.CAG.GFP.WPRE- and AAV8.CAG.hREP1.WPRE-injected mice were incubated in Fc block immediately after dissociation and were subsequently stained with cell surface markers. Antibody staining was performed in all retinae on ice for 20 min, with samples protected from light. Cells were subsequently washed with staining buffer prior to flow cytometric analysis. Retinal cells from AAV8.CAG.GFP.WPRE- and AAV8.CAG.hREP1.WPRE-injected mice were stained with 4′,6-diamidino-2-phenylindole (DAPI) (BioLegend, London, UK) 5 min prior to flow cytometric analysis.
The antibodies used in this study were as follows: CD45 Brilliant Violet 711 clone 30-F11 (0.5 μg/mL), CD45 PE clone 30-F11 (0.5 μg/mL), CD11b-APC clone M1/70 (0.25 μg/mL), CD3-Alexa Fluor 700 clone 17A2 (5 μg/mL), CD4 Brilliant Violet 785 clone RM4-5 (0.5 μg/mL), CD19 APC/Fire 750 clone 6D5 (2 μg/mL), NK-1.1 PE/Cyanine7 clone PK136 (1 μg/mL), and CD11c PerCP/Cyanine5.5 clone N418 (1 μg/mL) (all from BioLegend, London, UK).

Following gas plasma exposure, the microorganisms in the microwell plates were kept at room temperature in the dark for 16 h. In some conditions, 2 µL of catalase solution (Sigma-Aldrich, Taufkirchen, Germany) was added (final concentration: 20 µg/mL) either before or after gas plasma exposure of samples. Then, to each well, fixative was added (4% paraformaldehyde; Sigma-Aldrich) and incubated for 10 min in the dark. Then, to each well, 4′,6-diamidino-2-phenylindole (DAPI, final concentration 10 µM; BioLegend, Amsterdam, The Netherlands) was added, and the microorganisms were incubated for 15 min in the dark. All microorganisms contain ample amounts of DNA to which DAPI binds and becomes fluorescent. This way, microorganisms can be conveniently detected using flow cytometry. Next, the microplates were added to an autosampler of a CytoFLEX S flow cytometer (Beckman-Coulter, Krefeld, Germany) trigger through the forward scatter (488 nm laser diode) and collection of DAPI fluorescence via λ_ex 405 nm and λ_em 450 ± 45 nm. After mixing by the autosampler, 100µL of cell suspension was acquired from each well. The resulting .fcs (3.1 standard) data files were analyzed using Kaluza analysis software 2.1.3 (Beckman-Coulter).

Bone marrow single-cell suspensions were made by flushing femurs and tibiae with PBS + 2% fetal calf serum (FCS). All cell suspensions were treated with ACK buffer for red cell lysis. For flow cytometic analysis cell suspensions were stained with the appropriate combination of the following antibodies: anti-FceRI-PE (MAR-1; BioLegend); ant-CD49b-PE-Cy7 (DX5; Biolegend); anti-CD90.2-APC (30-H12; Biolegend); anti-CD19-APC (1D3; eBioscience); anti-IgM-PE-Cy7 (RMM-1; Biolegend); anti-IgD-eFluor450 (11-26; eBioscience); anti-CD93-PE (AA4.1; Biolegend); anti-CD2-FITC (RM2-5; BD Biosciences). Dead cells were excluded with Zombie UV Fixable viability dye (BioLegend). For cell cycle analysis and Nicoletti assay cells were fixed with the FOXP3/Transcription Factor Staining Buffer Set (eBioscience) and DNA was stained with 4,6 diamidino-2-phenylindole (DAPI; BioLegend). Flow cytometry was conducted using an LSRFortessa 5-laser (325; 405; 488; 561; 632) configuration (BD Biosciences). For FACS cells were collected using a MoFlo Astrios (Beckman Coulter) and sorted directly into Opti-MEM+ 10% FCS Media.