Cd4 fitc cd8 pe cd3 percp

The percentage of peripheral lymphocytes expressing TLR2 was measured. Blood samples of 50 µL with the following antibodies were used:□

CD19 FITC/CD3 PE;

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CD4 FITC/CD8 PE/CD3 PerCP;

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CD3 FITC/CD16 PE/CD56PE;

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TLR 2 PE/CD4FITC;

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TLR2PE/CD8FITC;

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TLR2PE/CD19FITC (BD Biosciences, San Jose, CA, USA).

Thereafter, the samples were treated with Lysing Buffer (BD Pharm Lyse) and PBS solution (Sigma Aldrich) for washing. We then collected the samples using a Cytoflex LX (Beckman Coulter) and analyzed the data using the Kaluza Analysis program. The data were evaluated with dot plots. Isotype controls (Biolegend) were used to determine unspecific binding.
We also calculated the percentages of the following: T helper cells (CD3+ CD4+), T cytotoxic cells (CD3+ CD8+), B lymphocytes (CD3− CD19+), natural killer cells (CD3− CD16+ CD56+) and natural killer T-like cells (CD3+ CD16+ CD56+). Using forward and lateral dispersions and a two-color fluorescence plot, we established the population of target cells. Gates were placed around individual cell populations to determine the relative CD45+ cell percentages. To determine the background signal and to exclude contamination and cell aggregates, FITC IgG1 κ and PE IgG1 κ were used as Isotype controls.

Routine analyses for absolute concentrations of CD3, CD4, and CD8 positive T cells and NK cells were evaluated by flow cytometric analyses with BD™ Trucount tubes containing fluorescent beads as an internal standard according to the manufacturer's instructions (BD Biosciences, San Jose, California). Immunolabeling with CD4 FITC/CD8 PE/CD3 PerCP and CD3 FITC/CD16+CD56 PE/CD45 PerCP from BD™ Biosciences were used. Samples were analyzed on Navios™ (Beckman Coulter, Miami, Florida), and Navios™ software was used for software analyses. Residual blood volume was used for immune phenotyping in a 2-tube, stain-lyse, multi-color flow cytometry panel developed for the study. For multi-fluorochrome staining, 100 μL of whole blood was labeled for TCRαβ-FITC, TCRγδ-PE, CD4-PerCP-Cy5.5, CD45RA-PE-Cy7, CD197-APC, CD45RO-APC-H7, HLA-DR-V450, CD3-V500, and CD8-BV605 for tube 1, and TCRVδ2-FITC, TCRγδ-PE, TCRVδ1-PE-Cy7, CD314-APC, CD16-APC-H7, CD56-V450, CD3-V500, and CD337-BV605 for tube 2. After 15 min incubation, red blood cells were lysed by the addition of 2 mL of lysing solution (EasyLyse™) followed by 10 min of dark incubation at room temperature. This was followed by a 5-min wash procedure (300 g) with 2 mL BD™ FacsFlow and resuspension in 300 mL BD ^TM FacsFlow prior to immediate acquisition.

We used the following antibodies in this study: NKG2C-APC, NKG2C-PerCp, NKG2A-PE (R&D); CD3-FITC, CD4-APC-Cy7, CD16-PerCP-Cy5.5, CD56-PE-Cy7, CD107a-APC-H7 (BD Biosciences, San Jose, CA, USA); IFN-γ-BV421 (Biolegend, San Diego, CA, USA); CD4-FITC/CD8-PE/CD3-PerCP (BD Biosciences, San Jose, CA, USA).