Cd69 pe

Flow cytometric analyses were performed using either fluorescently labeled or unlabeled mAbs followed by species-specific PE conjugates. Murine anti-human CD133 mAbs 293C3, AC133 and W6B3C1 were purchased from Miltenyi Biotec (Bergisch Gladbach, Germany). CD69-PE and CD107a-PE were from BD Pharmingen (San Diego, CA, USA), CD56-APC and CD14-PE/Cy7 from BioLegend (San Diego, CA, USA) and CD3-eFluor450 from eBioscience (San Diego, CA, USA). The goat anti-mouse PE conjugate was obtained from Dako (Glostrup, Denmark), the donkey anti-human PE conjugate was from Jackson ImmunoResearch (West Grove, PA, USA). The corresponding isotype controls were from BD Pharmingen (San Diego, CA, USA). Dead cells were excluded from analysis by 7-AAD (BioLegend; San Diego, CA, USA). Analysis was conducted using a FACS Canto II or FACS Fortessa (both BD Biosciences; Heidelberg, Germany). Specific fluorescence intensity (SFI) levels were calculated by dividing mean fluorescences obtained with a specific mAb by mean fluorescences obtained with the respective isotype control.

Cells were collected at different time points, washed with PBS, and centrifuged at 350g for 5 minutes. Cells were resuspended in FACs buffer (Thermo Fisher Scientific) and incubated in antibodies for 25 minutes in the dark at room temperature. Following incubation, cells were washed twice with FACs buffer and analyzed with a Cytoflex (Beckman Coulter). 10000 events were recorded per sample. To analyze T cell proliferation and activation within the PBMC cultures, first CD3+ cells were gated. For T cell activation CD25+ and CD69+ cells were gated within the CD3+ population. For T cell proliferation experiments a CD3 APC (BD Biosciences 555342) antibody was used. For T cell activation experiments CD3 APC (BD Biosciences 555342), CD69 PE (BD Biosciences 654975), and CD25 FITC (BD Biosciences 555431) antibodies were used. For T cell viability experiments CD3 APC (BD Biosciences 555342) and Propidium Iodide PE (Thermo Scientific BMS500PI) antibodies were used. For EV uptake experiments CD3 APC (BD Biosciences 555342), CD4 APC (Biolegend 317415), CD8 APC (Biolegend 344721) antibodies were used. Isotype antibodies used were: Anti IgG1κ PE (BD Biosciences 555749), Anti IgG1 FITC (Thermo Fisher GM4992), Anti IgG1κ APC (BD Biosciences 555751), Anti IgG2aκ APC (BD Biosciences 555576).

Fc-gamma receptors were blocked with a rat anti-mouse CD16/CD32 antibody (BD). Mouse-specific antibodies were the following: CD3-Brilliant violet 510, CD25-PercP Cy5.5, I-A/I-E-Brilliant violet 510, CD45R/B220-PE-Cy7, CD43-APC, CTLA-4-APC (all from BioLegend), CD4-APC-Cy7, CD69-PE, CD11c-PercP Cy5.5, CD19-APC-Cy7, CD40-FITC (all from BD Pharmingen), CD8α-PE-Cy7, FoxP3-FITC, CD86-APC, CD83-FITC, CD80-PE-Cy7, IgM-PercP-eFluor 710 (all from eBioscience).
During the experimental set-up, CD3 antibody was included in the staining panel (BV510 BioLegend clone 17A2) and used for the gating of T cells. The results obtained with gating on CD3+ CD4+ CD8- were similar to those obtained with gating on CD4+ CD8-. Due to the limitation in the maximum number of colours that can be discriminated with the FACS Canto II, CD3 staining was omitted in subsequent stainings.
Dead cells were excluded using the fixable viability dye-eFluor 450 (eBioscience), and intracellular staining was performed using the fixation/ permeabilization buffer set from eBioscience. Flow cytometry measurements were performed on a FACS Canto II instrument (BD) and the data were analyzed with FlowJo software (Tree Star).

To analyze immune infiltration, lung suspensions were stained for 30 min at 4°C using following directly conjugated antibodies: CD45 BV786 (BD Horizon, clone 30-F11); F4/80 BV650 (BD OptiBuild, clone: T45-2342); CD11c BV605 (BD Horizon, clone HL3); CD80 PE (BD Pharmingen, clone 16-10A1); MHC II BB700 (BD OptiBuild, clone 2G9); CD69 PE (BD Pharmingen, clone H1.2F3) and LIVE/DEAD FVS780 BD HorizonFixable Viability Stain 780). A purified rat anti-mouse CD16/CD32 MAb (eBiosciences, clone 93) was used to block non-specific binding to mouse Fc receptors.
The cells were analyzed using a FACS Celesta flow cytometer (BD Biosciences) and FlowJo software (TreeStar). All analyses were performed gating on CD45+ live cells after doublet exclusion.

Whole blood was drawn in EDTA tubes and the cells were incubated with fluorochrome-conjugated antibodies against human CD3-FITC, HLA-DR-FITC, CD45RA-FITC, CD8-PE, CD16+56-PE, CD69-PE, CD28-PE, CD3-PerCP, CD45-PerCP, CD45RO-PerCP-Cy5.5, CD4-PE-Cy7, CD25-PE-Cy7, CD25-APC, CD39-APC, CD62L-APC, CD8-APC-H7, CD4-APC-H7, CD3-Horizon V450, CD56-Horizon V450, all from BD Biosciences (San Jose, USA). Anti-Foxp3-PE was purchased from eBioscience (San Diego, USA) and Helios-Pacific Blue from Biolegend (San Diego, USA). Lymphocyte subpopulations from peripheral blood were measured by 4- or 7-color combinations. TruCount^™ tubes (BD Biosciences) were used for assessment of absolute numbers (cells/μl) of leukocytes (CD45), and major lymphocyte populations; T lymphocyte (CD3), T helper (CD4), T cytotoxic (CD8), B (CD19) and NK (CD16+CD56) cells, as described by the manufacturer. Absolute numbers of subpopulations were derived from the major populations. Proportions of major populations were given as % of lymphocytes and proportions of subpopulations were given as % of their parent population.