Percp anti mouse cd8

Twenty four weeks post final DNA immunization, the splenocytes were isolated from the immunized mice (n = 6 per group). CD4⁺ memory cells were stained with PerCP-Cy5.5-anti-mouse CD4 (Biolegend), FITC-anti-mouse CD44 (Biolegend), PE-anti-mouse CD62L (Biolegend), and APC-Cy7-anti-mouse CD25 (Biolegend). CD8⁺ memory cells were stained with PerCP-anti-mouse CD8 (Biolegend), PE-anti-mouse CD62L (Biolegend), and Pacific blue-anti-mouse CD45R (Biolegend) antibodies. The stained cells were fixed with Cytofix/Cytoperm Buffer™ (Becton Dickson) and then analyzed with a FACS Canto II flow cytometer with FACSDiva software.

HEK293T cells stained Alexa Fluor 647-conjugated annexin V, 1:50 (BioLegend) and propidium iodide (1 µg/mL) in DMEM (Nacalai Tesque, 08459-64) with 10 mM HEPES on room temperature for 15 min. Samples were filtered through a 37 µm mesh prior to cytometry analysis.
Splenocytes were collected from adult and P9, P5 mice, and P5 interspecies chimeras by mashing the spleen between glass slides and chilling it in PBS(–). Splenocytes were stained with APC anti-mouse CD45 (1:50, Biolegend 100516), PE anti-mouse CD3ε (1:50, Biolegend 100308), Alexa Fluor 488 anti-mouse CD3ε (1:50, Biolegend 100321), APC anti-mouse CD4 (1:50, Biolegend 100516), PerCP anti-mouse CD8 (1:50, Biolegend 100732), APC anti-rat CD45 (1:50, Biolegend 202212), PE anti-rat CD3ε (1:50, Biolegend 201412), APC anti-rat CD4 (1:50, Invitrogen 17-0040-82), and PerCP anti-rat CD8 (1:50, Biolegend 201712) in 50 µL of 0.1% BSA/PBS at 4 °C for 30 min. The samples were resuspended in 500 µL of 0.1% BSA/PBS and filtered through a 37 µm mesh prior to flow cytometry analysis.
Flow cytometry was conducted using a BD Accuri C6 flow cytometer (BD Biosciences), and data were analyzed using FlowJo software (Becton Dickinson).

Antigen-specific memory CD4⁺ and CD8⁺ T cell immune responses were assessed using an intracellular cytokine staining (ICS) assay [53 (link), 54 (link)]. Briefly, freshly isolated splenocytes from the NC and mRNA vaccine-immunized groups on day 42, along with SARS-CoV-2 S protein peptide pools (2 µg/ml for each peptide), were cultured in 24-well plates at a density of 2 × 10⁶ cells/well. After a 2-hour incubation, monensin (YEASEN) was added to each well to inhibit cytokine secretion. Twelve hours later, the cells were harvested and stained for 40 min with PE anti-mouse CD4, PerCP anti-mouse CD8, Alexa Fluor 700 anti-mouse MHC II, BV421 anti-mouse B220, BV510 anti-mouse CD44, BV711 anti-mouse CD3 (Biolegend), and Pacific Orange for live/dead cells (Thermo Fisher). Subsequently, the cells were fixed with fixation buffer for 20 min and permeabilized in 1× permeabilization buffer (Biolegend). Intracellular staining was performed using FITC anti-mouse IFN-γ, PE/Cyanine7 anti-mouse TNF-α (Bioss), and APC anti-mouse IL-4 (Biolegend). Following three washes with PBS, the cells were analyzed by flow cytometry using a NovoCyteTM system (Eisen).