BD Cytofix/Cytoperm Method

Th17 culture analyzed using Human Th1/Th2/Th17 Phenotyping Kit
Flow cytometric analysis of a Th17 culture using the Human Th1/Th2/Th17 Phenotyping Kit
Human peripheral blood mononuclear cells (PBMCs) were stimulated for 13 days with anti-CD3 and anti-CD28 monoclonal antibodies in the presence of recombinant IL-6, TGF-β, IL-1β, and IL-23, as well as anti–IFN-γ and anti–IL-4 neutralizing antibodies. Harvested cells were stained according to the Human Th1/Th2/Th17 Phenotyping Kit (Cat. No. 560751) instructions and analyzed on a BD™ LSR II flow cytometer. Dot plots are based on a CD4+ lymphocyte gate.
Intracellular cytokine analysis in polarized Th cell subsets
Intracellular staining of cytokines in cultured Th1, Th2, Th9, Th17, and iTreg cells

PBMCs were cultured for 14 days in Th1, Th2, Th9, Th17, and iTreg polarization conditions and restimulated with PMA/ionomycin for 5 hours in the presence of BD GolgiStop™ protein transport inhibitor (monensin, Cat. No. 554724). The cells were then fixed with BD Cytofix™ fixation buffer (Cat. No. 554655), permeabilized with BD Perm/Wash™ buffer (Cat. No. 554723), and stained with BD Pharmingen™ antibodies against CD4 and intracellular cytokines IL-9, IFN-γ, IL-4, or IL-17A. The dot plots were derived from a lymphocyte gate. Flow cytometry was performed on a BD LSR II system.

Normal human PBMCs were isolated from whole blood by Ficoll-Paque™ Plus, and stimulated and expanded with plate-bound anti-CD3 (10 µg/mL) and soluble anti-human CD28 (1 µg/mL) plus recombinant cytokine IL-2 (10 ng/mL) in the presence of:

  • TH1, IL-12 (50 ng/mL) plus neutralizing mAb to human IL-4 (10 µg/mL)
  • TH2, IL-4 (50 ng/mL) plus neutralizing mAb to IFN-γ (10 µg/mL)
  • TH17, IL-6 (50 ng/mL), IL-1β (10 ng/mL), IL-23 (10 ng/mL), TGF-β (10 ng/mL) plus neutralizing mAb to IFN-γ (10 µg/mL) and IL-4 (10 µg/mL)
  • iTreg, TGF-β (10 ng/mL) plus neutralizing mAb to IFN-γ (10 µg/mL) and IL-4 (10 µg/mL)
  • TH9, IL-4 (50 ng/mL), TGF-β (10 ng/mL), and neutralizing mAb to IFN-γ (10 µg/mL)
Intracellular IL-17A and IL-17F staining
IL-17A and IL-17F staining using a BD Cytofix/Cytoperm™ protocol
Human cells polarized toward a Th17 phenotype were characterized for IL-17A and IL-17F expression using the BD Cytofix/Cytoperm protocol. Cells were cultured in the presence of Th17-polarizing cytokines and with a protein transport inhibitor, such as BD GolgiStop (monensin, Cat. No. 554724) or BD GolgiPlug™ (brefeldin A, Cat. No. 555029), to prevent secretion and thus allow accumulation of the cytokine inside the cell. Cells were fixed and permeabilized with BD Cytofix/Cytoperm fixation/permeabilization solution (Cat. No. 554722) to allow fluorescent antibodies to enter the cell and bind to their target cytokines. Cells were then stained with antibodies to CD4, IL-17A (Alexa Fluor® 647-conjugated, Cat. No. 560437), and IL-17F (PE-conjugated, Cat. No. 561198) and then detected by multicolor flow cytometry.
Intracellular cytokines in Th17 cells (time course)
Representative data from a time course of Th17-polarized cells comparing levels of IL-17A with CD4, IFN-γ, and IL-4.
Human PBMCs were stimulated for 14 days with anti-CD3 and anti-CD28 monoclonal antibodies in the presence of recombinant IL-6, TGF-β, IL-1β, and IL-23, as well as anti–IFN-γ and anti–IL-4 neutralizing antibodies. Cells were treated with BD GolgiStop (monensin) inhibitor, fixed and permeabilized with BD Cytofix/Cytoperm buffer, and then stained with BD Pharmingen antibodies against the indicated cytokines. At 6 days there were significant numbers of cells expressing IL-17A, with numbers of cells increasing at day 10 and then leveling off.



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