Anti granzyme b fitc

To analyze cell surface proteins levels, 5 × 10⁵ lymphocytes were labeled with the following mAntibodies: anti-B220 FITC, anti-CD4 PE, anti-IFNγ PE (BD Pharmingen™), anti-CD122 PE, anti-CD127 PE, anti-CD25 APC, anti-CD25.AF488, anti-CD3 APC, anti-CD44 FITC, anti-CD62L PE, anti-CD62L PErCP.Cy5.5, anti-CD8 PerCP.Cy5.5, anti-Granzyme B FITC, anti-IgG2a K FITC, anti-IgG2a K PE, anti-Klrg1 FITC (all eBioscience™). For intracellular staining, 1 × 10⁶ cells/ml were stimulated in vitro for 6 h with 10 nM of Phorbol 12-myristate 13-acetate (PMA) plus 1 μM of ionomycin (both from Calbiochem^®). Brefeldin A (1:1000; BD Pharmingen™) was added to the culture for the last 2 h. Cells were harvested and stained with anti-CD44 APC and anti-CD62L PErCP.Cy5.5 antibodies. Then, the cells were fixed, permeabilized, and stained with anti-Granzyme B FITC, anti-IFN-γ PE or anti-IL-2 PE antibodies and analyzed by flow cytometry on a FACSCalibur. For the memory subtype analysis, day 10 memory cells generated in vitro were sorted using a MoFlo Cell Sorter (Beckman Coulter, Inc) based on the CD62L level. All the flow cytometry data were analyzed using FlowJo^® software.

The mice were sacrificed and the blood and splenocytes were isolated and resuspended in 100 μL PBS. FcR‐blocking reagent (BD PharMingen, San Diego, USA) was used to reduce nonspecific binding of antibodies. To detect antigen‐presenting capacity, cells were divided in two tubes where one was incubated with anti‐CD11c PE, anti‐HLA FITC and anti‐CD80 APC antibodies (all from Biolegend, San Diego, USA) for 20 minutes on ice and a second tube was incubated with anti‐CD11b PE and anti‐CD80 APC antibodies (Biolegend). Cytotoxic T cell detection was performed by staining the cells with anti‐CD8 PE (Biolegend) and anti‐GranzymeB FITC (eBioscience, USA) antibodies. Regulatory T cell detection was performed by staining the cells with anti‐CD4 Flu647, anti‐CD25 PE and anti‐Foxp3 FITC antibodies (Biolegend). Blood T cell differentiation was performed by staining the cells with anti‐CD4 FITC, anti‐CD44 APC, anti‐CD8 PE antibodies (Biolegend). True‐Nuclear Transcription Factor Buffer Set (Biolegend) was used for intracellular transcription factors staining. Briefly, cells were incubated with 1 mL fixing buffer for 20 minutes on ice and further incubation in the perm buffer for 30 minutes. Antibodies were diluted in perm buffer and incubated with the cells on ice for 30 minutes.