Granzyme b fitc

In-vitro cytotoxicity was assessed using K562 tumor targets at a 10:1 E:T ratio as previously described (43 (link)). Intracellular granzyme B and CD107a expression was measured by flow cytometry as previously described (43 (link)). Briefly, PBMCs were mixed with RPMI1640 medium or target cells at a ratio of 10:1 in the absence of exogenous cytokines in medium. For CD107a expression, anti-CD107a-FITC (Miltenyi Biotech) was added to each well and incubated for 1 hour at 37°C. After 1 hour, brefeldin A (Golgi Plug; BD Biosciences) was added to each well, and the cells were incubated for additional 3 hours. Cells were then washed, fixed, permeabilized using Cytofix/Cytoperm reagent kit (BD Biosciences), and resuspended in staining buffer containing anti-CD56-PE-Vio770 (Miltenyi Biotech). For Granzyme B levels, cells were incubated for 4 hours, washed, fixed, permeabilized using a Cytofix/Cytoperm reagent kit (BD Biosciences), and resuspended in staining buffer containing anti-CD56-PE-Vio770 (Miltenyi Biotech) and granzyme B-FITC (Miltenyi Biotech).

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).