Pe cy7 conjugated anti cd4 clone sk3

The number of circulating blood Th cells expressing TLR2 (CD4⁺CD282⁺) was assessed by flow cytometry using labelled monoclonal antibodies (BD, USA): APC-H7-conjugated anti-СD45 (clone 2D1, mouse IgG1κ), PE-Cy7-conjugated anti-CD4 (clone SK3, mouse IgG1κ), and Alexa Fluor 488-conjugated anti-CD282 (clone 11G7, mouse IgG1κ). The data were analyzed by the FACSDiva software (BD, USA). At least 100,000 events were acquired for each sample. The results were expressed as the percentage of the CD4⁺ lymphocyte population.

Following 3 days co-incubation of yeast stimulated DCs and naive T cells, cells were washed using cold CellWash (BD Biosciences, Erembodegem, Belgium) and resuspended in cold Stain Buffer (BD Biosciences, Erembodegem, Belgium). For surface staining, cells were incubated with PE-Cy7-conjugated anti-CD4 (clone SK3) and PerCP-Cy5.5-conjugated anti-CD25 (clone M-A251) or appropriate nonspecific isotype matched controls (all from BD Biosciences, Erembodegem, Belgium) for 30 min on ice protected from light. For intracellular staining, surface-stained cells were fixed and permeabilized using Human Foxp3 Buffer Set (BD Biosciences, Erembodegem, Belgium), incubated with PE-conjugated anti-Foxp3 (clone 259D/C7) or an appropriate nonspecific isotype matched control (both from BD Biosciences, Erembodegem, Belgium), and washed twice before analysis. Stained cells were acquired on an LSRFortessa flow cytometer (BD Biosciences, San Jose, CA, USA) using FACSDiva software (BD Biosciences, San Jose, CA, USA). Specific gates and quadrants were defined based on background staining of isotype controls. At least 10,000 cells were analyzed for each sample.