2.4. Flow cytometric analyses

To detect Phl p 5⁺/GFP-expressing CD19⁺ B cells, flow cytometric analyses were performed. The percentage of Phl p 5⁺ cells was calculated by subtracting the number of positive events observed in control staining from those quadrants containing Phl p 5⁺ and Phl p 5^- cells expressing a CD19⁺ marker and by dividing the net percentage of Phl p 5⁺ cells by the total net percentage of Phl p 5⁺ plus Phl p 5^- CD19⁺ B cells as described (33). Phl p 5-specific polyclonal antibodies were purified from rabbit serum raised against rPhl p 5 on a HiTrap Protein G HP antibody purification column (Merck, Darmstadt, Germany) according to the manufacturer’s instructions. Polyclonal anti-Phl p 5 IgG was used as the primary antibody and developed by counterstaining with PE donkey anti-rabbit IgG. 7AAD (Viability Staining Solution, BioLegend, San Diego, USA) was used to exclude dead cells. CD19-APC-Cy7 (clone: 6D5) and CD11b-PE (clone: M1/70) were purchased from BioLegend (San Diego, USA). CD4-PE-Cy7 (clone: GK1.5), CD45.2-eFluor500 (clone: 104), and CD3- eFluor450 (clone: 17A2) were purchased from Thermo Fisher Scientific (MA, USA). CD8-APC (clone: 53-6-7) and CD45.2-PerCP-Cy5.5 (clone: 104) were purchased from BD Biosciences (San Diego, CA, USA). Flow cytometric analyses were performed using a BD FACS Canto II (BD Biosciences, San Diego, CA, USA), and data were analyzed using FlowJo software version 10.7.2.