Anti mouse cd3a pe

Following immunization, the magnitude of the splenic T cell differentiation was assessed by fluorescence-activated cell sorting assay (FACS). Briefly, 1 × 10⁵ viable cells were seeded in 96-well plates, stimulated with purified NP-Gn/Gc-epitope, NP, Gn/Gc, and NS recombinant proteins for 48 h, and incubated at 37 °C. After 72 h, cells were stained with anti-mouse CD3a-PE (Miltenyi Biotec, Bergisch-Gladbach, Germany), CD4-perCPvio700 (Miltenyi Biotec), and CD8a-FITC (Miltenyi Biotec) antibodies (8 µg/mL) for 30 min at 4 °C in the dark. Finally, cells were washed with FACS running buffer (Miltenyi Biotec), and the T-cell populations CD3⁺CD4⁺ and CD3⁺CD8⁺ were gated from the CD3⁺ population and analyzed using the MacsQuant analysis system (Miltenyi Biotec).

Following vaccination, the magnitude of the differentiation of splenic T cells was assessed using fluorescence-activated cell sorting (FACS), as previously described. Briefly, the mice were euthanized, and spleens were aseptically isolated at day 10 post-immunization. The prepared splenocytes (1 × 10⁶ cells/mL) were incubated with a mixture of fluorescent-labeled antibodies, such as anti-mouse CD3a-PE, anti-mouse CD4-perCP-vio700, and anti-mouse CD8a-FITC (Miltenyi Biotec, Bergisch Gladbach, Germany) for 15 min at 4 °C in the dark. The samples were washed twice with FACS buffer (Miltenyi Biotec) and resuspended in 200 μL of FACS buffer. The cells were then examined with the MACSQuant^® Analyzer (Miltenyi Biotec). The results were further analyzed using FlowJo Software (Tree Star Inc., CA, USA).

An alteration in T cell subsets of the immunized mice was assessed using fluorescence-activated cell sorting (FACS) (Jawale and Lee, 2014 (link)). Spleen cells were isolated from the immunized and non-immunized mice at day 7 PI and 1 × 10⁶ of the cells were seeded into a 96-well cell culture plate. The splenic cells were stained with the surface markers containing anti-mouse CD3a-PE and anti-mouse CD4-perCP-vio700 (Miltenyi Biotec, Bergisch Gladbach, Germany). After the stained cells were washed twice in FACS buffer (Miltenyi Biotec, Bergisch Gladbach, Germany), the stained CD3+ and CD3+CD4+ splenic cells were sorted with an MACSQuant^® Analyzer (Miltenyi Biotec, Bergisch Gladbach, Germany). The altered FACS profiles of CD3+ and CD3+CD4+ splenic T cell subsets in the immunized mice were analyzed in comparison to those from non-immunized mice.