Anti mouse igg1 apc

PBMC were stained with anti-CD14 mAb conjugated to Fluorescein isothiocyanate (FITC) and–CD16 mAb conjugated to Allophycocyanin (APC) (both purchased from BD Biosciences, San Jose, CA, USA). To set the gates, control isotype antibodies were employed, an anti-mouse Ig kappa chain-FITC (BD Pharmingen), and an anti-mouse IgG1-APC (BioLegend). Moreover, monocytes were gated on the basis of their size (forward scatter) and complexity (side scatter), and the monocyte subsets were identified by the levels of expression of CD14 and CD16. Fifty thousand events were acquired for each sample on a FACSCanto II (two-laser, six-color configuration) with the FACSDiva 6.1.3 software (BD Biosciences). The results are shown as percentage of each subpopulation. Nomenclature of monocyte subsets followed the recommendations of the Nomenclature Committee of the International Union of Immunological Societies. We carried out daily routine quality control tests with Cytometer Setup & Tracking Beads (BD Biosciences) in accordance with the manufacturer’s instructions.

A modified version of our previously published serological assay was used^{54 (link)}, in which stably transduced HEK 293 T cells express the antigen of interest. To analyse quantities of antigen-specific antibodies, 5 × 10⁵ HEK 293 T cells producing SARS-CoV-2 spike or nucleocapsid were incubated for 20 min at 4 °C with the respective biological sample diluted in 100 µl FACS-PBS (PBS with 0.5% BSA and 1 mM sodium azide) to bind to spike protein on the surface, or in 100 µl permeabilization buffer (0.5% saponin in FACS-PBS) to bind to intracellular nucleocapsid protein. After washing with 200 µl buffer, specific antibodies were bound with polyclonal anti-mouse Ig-FITC (1:300, 4 °C, 20 min incubation; BD Biosciences), anti-mouse IgG1-APC (1:300, clone X56, Biolegend), or anti-mouse IgG2a-FITC (1:300, clone R19-15, BD Biosciences). After further washing, samples were measured on an AttuneNxt (ThermoFisher) and analysed using FlowJo software (Tree Star Inc.).