BD FastImmune cytokine detection system

Typical CD4 IFN-γ responses to three different antigens measured using the BD FastImmune™ cytokine system

Heparinized whole blood from seropositive individuals was restimulated with antigen and costimulatory antibodies (CD28 and CD49d) in the presence of the secretion inhibitor brefeldin A, fixed using BD FACS™ lysing solution (Cat. No. 347691), then washed and permeabilized with BD FACS™ Permeabilizing Solution 2 (Cat. No. 340973). Samples were analyzed using the BD FastImmune CD4 intracellular IFN-γ detection kit (Cat. No. 340970). Typical frequencies of IFN-γ–producing cells are shown for antigen-restimulated (+Ag) and unstimulated (-Ag) samples.

Simultaneous detection of TNF, IFN-γ, and IL-2 in CD4⁺ and CD8⁺ T cells

Cryopreserved peripheral blood mononuclear cells (PBMCs) were stimulated with SEB (1 µg/mL) in the presence of brefeldin A for 6 hours. Activated cells were lysed and fixed using BD FACS lysing solution (Cat. No. 347691), then washed and permeabilized using BD FACS Permeabilizing Solution 2 (Cat. No. 340973). Cells were stained with a 6-color antibody panel including BD FastImmune cytokine antibodies [TNF FITC (Cat. No. 340511), IFN-γ PE (Cat. No. 340452), IL-2 APC (Cat. No. 341116), CD8 PerCP-Cy™5.5 (Cat. No. 341051), CD4 PE-Cy™7 (Cat. No. 348789), and CD3 BD Horizon™ V450] and analyzed on a BD FACSCanto™ II flow cytometer. Dot plots are based on a lymphocyte (FSC/SSC) and CD3⁺ gate.

Analysis of cytokine expression in CD4⁺ and CD8⁺ T-cell subsets using a BD FastImmune protocol

Human PBMCs were isolated from a healthy donor using CPT tubes. Cells were stimulated for 6 hours with SEB (1 µg/mL), in the presence of brefeldin A. Activated cells were then washed and stained with CD3 APC-H7 (Cat. No. 641397), CD4 PerCP-Cy™5.5 (Cat. No. 341654), CD8 BD Horizon™ V500 (Cat. No. 560774), CD28 APC (Cat. No. 559770), CCR7 PE (Cat. No. 552176), CD45RA PE-Cy™7 (Cat. No. 337167). Cells were then fixed by incubation with BD FACS lysing solution (Cat. No. 347691), washed, and permeabilized using BD FACS Permeabilizing Solution 2 (Cat. No. 340973). Cells were then washed twice, stained with IFN-γ BD Horizon V450 (Cat. No. 560371), IL-2 FITC (Cat. No. 340448), TNF Alexa Fluor® 700 (Cat. No. 557996), and analyzed on a special order BD™ LSR II cell analyzer. Dot plots are based on a lymphocyte (FSC/SSC) and CD3⁺ gate.

Analysis of cytokine expression in naïve, memory, and effector T-cell subsets using a BD FastImmune protocol

Human PBMCs were isolated from a healthy donor using CPT tubes. Cells were stimulated for 6 hours with SEB (1 µg/mL), in the presence of brefeldin A. Activated cells were then washed and stained with CD3 APC-H7 (Cat. No. 641397), CD4 PerCP-Cy5.5 (Cat. No. 341654), CD8 BD Horizon™ V500 (Cat. No. 560774), CD28 APC (Cat. No. 559770), CCR7 PE (Cat. No. 552176), CD45RA PE-Cy7 (Cat. No. 337167). Cells were then fixed by incubation with BD FACS lysing solution (Cat. No. 347691), washed, and permeabilized using BD FACS Permeabilizing Solution 2 (Cat. No. 340973). Cells were then washed twice, stained with IFN-γ BD Horizon V450 (Cat. No. 560371), IL-2 FITC (Cat. No. 340448), TNF Alexa Fluor® 700 (Cat. No. 557996), and analyzed on a special order BD LSR II cell analyzer. Dot plots are based on a lymphocyte (FSC/SSC), CD3⁺ and CD4⁺ gate.