Cyan adp 9 color analyzer

Serum from immunized mice was prepared from whole blood, further purified by Protein A affinity chromatography and isolated polyclonal IgG antibodies adjusted to a concentration of approximately 2 mg*ml⁻¹. H. pylori J99 were CFDA-SE labeled, washed and blocked with heat inactivated FCS for 15 minutes at 37 °C under shaking. Subsequently, bacteria were washed with PBS with 0.5% (w/v) BSA, adjusted to an OD₆₀₀ of 0.5 and 100 µl seeded into a round bottom 96-well cell culture plate. Purified antibodies were diluted in PBS with 0.5% (w/v) BSA. Bacteria were pelleted by centrifugation, the supernatant discarded and the pellet resuspended with 100 µL the corresponding primary antibody dilution and incubated for 30 min on ice. After washing twice, the eFlour^®660 labeled secondary anti-mouse IgG1-HRP conjugate (eBioscience) was added and incubated for 30 minutes on ice. After washing twice, bacteria were fixed with PBS with 1% PFA, filtered and analyzed by flow cytometry on a CyAN ADP 9 color analyzer (Beckman Coulter), gating on CFSE positive cells. The experiment was controlled by antibodies derived from the following immunizations: HPA (UniProt ID B5Z7F9), an outer membrane protein of H. pylori; HPG (UniProt ID O25743), a protein located in the cytoplasm and inside of outer membrane vesicles; CT without antigen. Data were analyzed with the FloJo X software (Treestar).

Staining with anti-HLA-A2-PE (clone BB7.2, BD Biosciences, cat. no. 558570), anti-HLA-A2-FITC (clone BB7.2, BD Biosciences, cat. no. 551285), anti-CD4-PerCP (clone SK3, BD Biosciences, cat. no. 345770), and/or anti-CD25-PE (clone 4E3, Miltenyi Biotec; cat. no. 130-091-024) was performed in the dark with antibody dilutions in FACS buffer (PBS/0.5% HSA) for 15 min at 20°C or 30 min at 4°C. Cells were washed once with PBS, resuspended, and measured in FACS buffer. For intracellular FOXP3 staining, cells were first stained with surface antibodies as described earlier, followed by fixable viability dye-eFlour780 (ebioscience, cat. no. 65-0865-14) staining enabling gating on live cells. Subsequently, fixation, permeabilization, and intracellular staining was performed with the Foxp3 Staining Buffer Set (ebioscience, cat. no. 00-5523-00) using anti-FOXP3-APC (clone 236A/E7, ebioscience, cat. no. 17-4777-42) or equally concentrated mIgG1κ APC isotype control (clone P3.6.2.8.1, ebioscience, cat. no. 17-4714-42) antibodies. Acquisition was performed on a CyAn ADP 9 Color Analyzer (Beckman Coulter), and parameter compensation was performed automatically with the CyAn software (Summit) tool using single stained samples containing positive cells. Flow cytometry data were analyzed using the FlowJo software (Tree Star).

Staining with anti‐HLA‐A2‐PE (clone BB7.2, BD Biosciences), anti‐HLA‐A2‐FITC (clone BB7.2, BD Biosciences), anti‐CD4‐PErCP (clone SK3, BD Biosciences), anti‐CD4‐PE (clone #11830, R&D systems), anti‐CD25‐PE (clone 4E3, Miltenyi Biotec), and/or anti‐CD3‐PE‐Vio770 (clone BW264/56, Miltenyi Biotec) was performed in the dark with Ab diluted in FACS buffer (PBS with 0.5% HSA) for 15 min at 20°C or 30 min at 4°C. Cells were washed once with PBS, resuspended, and measured in FACS buffer. Where noted, following surface staining, viability staining was performed with Fixable Viability Dye eFluor 780 (eBioscience) according to the manufacturer's instruction (without subsequent fixation). Live cells were gated via fsc/ssc and, where applicable, on viability dye‐negative cells. Backgating of viability dye positive and negative populations confirmed that dead cells could be completely gated out by fsc/ssc with the purified CD4 T cells used. Acquisition was performed on a CyAn ADP 9 Color Analyzer (Beckman Coulter) or BD FACSVerse (BD Biosciences), and automatic parameter compensation was performed automatically with the CyAn software (Summit) tool or with FlowJo utilizing single stained control samples. Flow cytometry data were analyzed using FlowJo software (Tree Star) version 10.4.1.