Pacific orange for live dead cells

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).

The kinetics of S-specific memory B cell responses were determined according to previously described methods [55 (link), 56 ]. Freshly isolated splenocytes from the NC group and five mRNA vaccine-immunized groups at day 84 were initially stained with AVI tag-labeled S protein (Vazyme) for 30 min at 4 °C. After three washes with PBS, the cells underwent further staining with FITC anti-mouse CD16, PE anti-mouse CD4, PE/Cyanine5-conjugated streptavidin (binding to the AVI tag), PerCP/Cyanine5.5 anti-mouse IgM, PE/Cyanine7 anti-mouse CD20, APC/Cyanine7 anti-mouse CD14, Pacific Blue anti-mouse CD19, Qdot655 anti-mouse IgD, Qdot705 anti-mouse CD3 (Biolegend), and Pacific Orange for live/dead cells (Thermo Fisher). Subsequent to three additional PBS washes, the cells were analyzed using NovoCyteTM flow cytometry (Eisen).