Efluor 450 conjugated anti cd3

Subsequent to isolation of splenic lymphocytes with Lymphocyte Gradient Separation medium (Mediatech Inc, Herndon, VA), lymphocytes were stimulated with 10 ng/mL PMA, 1 μg/mL ionomycin, and 10 μg/mL Brefeldin A for 5 hours. Surface staining was performed before permeabilization using perm/wash buffer (BD Biosciences, San Diego, CA). Permeabilized cells were subsequently incubated with intracellular-targeting antibodies. Efluor 450-conjugated anti-CD3, FITC-conjugated anti-CD4, Percp-Cy5.5-conjugated anti-CD8a, APC-conjugated anti-CD25, PE-conjugated anti-FOXP3, Percp-Cy5.5-conjugated IL-17, and PE-conjugated IFN-γ were purchased from eBioscience. The BD LSR II with BD FACSDiva software flow cytometry system and FlowLogic software (Inivai, VIC, Australia) were used to analyze data.

Fresh heparinized whole blood from healthy donors was utilized for neutrophil, CD14, and CD2 purifications. Whole blood was lysed (155 mM NH₄Cl, 10 mM KHCO₃, and 0.1 mM EDTA in distilled water) for 10 min at RT and washed twice for 10 min at 300 × g. CD66b⁺ neutrophils were isolated by positive selection using the CD66abce Microbead kit (Miltenyi Biotec) according to manufacturer’s protocol. Purity was assessed by flow cytometry using BV421-conjugated anti-CD66b and APC-conjugated anti-CD14 (BD Biosciences). The purity of the enriched population was >98%. CD14⁺ and CD2⁺ cells were positively isolated from the PBMC fraction obtained by Ficoll-Hypaque Plus gradient separation (GE Healthcare, France), using CD14 and CD2 Microbeads (Miltenyi Biotec), according to manufacturer’s protocol. Briefly, CD14⁺-labeled cells were first enriched on positive selection columns and the depleted fraction containing total lymphocytes was washed and stained with CD2 Microbeads and isolated on positive selection columns. Purity of CD14 and CD2 enriched fractions was assessed by flow cytometry using APC-conjugated anti-CD14 (BD Biosciences) and efluor450-conjugated anti-CD3 (Ebioscience, France) and was >98%. Viability of the samples following each purification procedure was assessed by Trypan blue staining and was >97% for all experiments.

Isolated lymphocytes were stained with cell surface antibodies. Efluor 450-conjugated anti-CD3, APC-conjugated anti-CD19, Efluor450-conjugated anti-CD4, and FITC-conjugated anti-CD8a were purchased from eBioscience. The BD LSR II with BD FACSDiva software was used to analyze data.