Cd69 pacific blue

Cells were initially blocked with anti-mouse CD16/32 (eBioscience) then stained with fluorochrome-conjugated antibodies following our previous procedures (30 (link)). Briefly, after blocking of Fc receptors, cells were stained with fluorochrome-conjugated antibodies on ice in the dark for 15 min. Exclusion of dead cells was achieved with Zombie Aqua (BioLegend) staining. For quantification of T cells in total splenocytes, the following anti-mouse antibodies were used: CD3-APC, CD4-PE-Cy7, CD8-PerCP-Cy5.5, CD44-PE, CD62L-APC-Cy7, and CD69-Pacific blue (BioLegend). The same panel of antibodies were used for in-vitro activated splenocytes. For analysis of apoptosis, FITC-Annexin V Apoptosis Detection Kit (BioLegend) was used together with propidium iodide according to the manufacturer’s procedures. Splenic Gr-1+ cells in the CD11b+CD11c− myeloid population were analyzed using the following anti-mouse antibodies: CD11c-APC, CD11b-PerCP-Cy5, PD-L1-PE-Cy7 (BioLegend), and Gr-1-V540 (BD Bioscience). Analysis was performed with a BD FACSAria II flow cytometer (BD Biosciences). Flow cytometry data were analyzed with FlowJo.

Experimentally infected monocytes were coincubated with CD8⁺ T cells from the same, seropositive donor overnight in the presence of CD107a Alexa Fluor 647, 5 μg/ml brefeldin A, and 2 μM monensin (all from BioLegend) at 37°C. CD8⁺ T cells were harvested and washed and then stained with a combination of surface antibodies (CD3 brilliant violet 650, CD14 brilliant violet 510, and CD19 brilliant violet 510; BioLegend) and LIVE/DEAD Fixable Aqua Dead cell stain (Invitrogen) at 4°C. Cells were fixed and permeabilized using FIX & PERM (ADG, Kaumberg, Austria) and stained intracellularly with antibodies (CD69 Pacific Blue, 4-1BB phycoerythrin [PE]-Cy5, CD8 brilliant violet 570, and granzyme A FITC [BioLegend]; granzyme B FITC [Miltenyi Biotec, Inc.]; granzyme K FITC [Santa Cruz Biotechnology, TX, USA]; TNF-α brilliant ultraviolet 395 and IFN-γ brilliant violet 786). Responding CD8⁺ T cell populations were identified by the expression of CD69 and 4-1BB at levels above the background, and expression levels of CD107a, TNF-α, and IFN-γ were then measured. In all cases, cell doublets, monocytes, B cells, and dead cells were eliminated from the analyzed populations.
To analyze differentiation markers, monocytes were labeled with anti-CD14 and anti-CD83 antibodies (BioLegend) (both allophycocyanin [APC] conjugated).

Cells were initially blocked with anti-mouse CD16/32 (eBioscience) then stained with fluorochrome-conjugated antibodies following our previously published procedures (55 (link)). Zombie Aqua (BioLegend) staining was performed to exclude dead cells. For quantification of B cells in total splenocytes, the following anti-mouse antibodies were used: CD19-Pacific blue, CD27-PE, CD138-APC-Cy7, CD44-PerCP-Cy5.5, IgD-PE-Cy7, GL7-AF647. For splenic T cells, CD3-APC, CD4-FITC, CD8-PE, CD44-PerCP-Cy5.5, CD62L-APC-Cy7, CD69-Pacific blue, CXCR5-PerCP-Cy5.5, and PD-1-APC-Cy7 (BioLegend) were used. For myeloid cell analysis, the following anti-mouse antibodies were used: CD11b-PE, CD11c-PerCP-Cy5, F4/80-PE-Cy7 (BioLegend), Gr1-V540 (BD Bioscience). Analysis was performed with a BD FACSAria II flow cytometer (BD Biosciences). Flow cytometry data were analyzed with FlowJo.