Pe conjugated anti ifn γ

Leukocytes were prepared from lymph nodes draining the inflamed joint, spleen, and thymus of NI and CIA mice. Synovial cells were harvested by digesting synovial tissues with collagenase. The cells were incubated with primary antibodies for 15 min at 4°C. Antibodies against CD4, CD8, CD44, Foxp3, B220, and CD11c were purchased from eBioscience; the anti-CD62L antibody was from BD Pharmingen. Nonspecific staining was ascertained using isotype-matched control antibodies. T_H cells were fixed in fixation/permeabilization buffer for 30 min; resuspended in 100 µl of permeabilization buffer; incubated for 30 min at 4°C with Alexa 488- or phycoerythrin (PE)-conjugated anti-IFNγ (eBioscience), fluorescein isothiocyanate (FITC)-conjugated anti-IL4, PE-conjugated anti-IL17A or isotype control antibodies (eBioscience); and analyzed by flow cytometry using EPICS XL and EXPO32 software (Beckman Coulter).

For intracellular IFN-γ ⁄ IL-4 ⁄ IL-17 staining and detection, 2 × 10⁶ splenocytes, lymphocytes, or liver cells from schistosome-infected or normal mice were surface stained with anti-CD3-APC mAbs (eBioscience, San Diego, CA) and anti-CD4-FITC mAbs for 30 minutes. Cells were washed, fixed and permeabilized with Cytofix/Cytoperm buffer (BD Pharmingen) for 40 minutes and then intracellularly stained with PE-conjugated anti-IFN-γ, anti-IL-4 or anti-IL-17 respectively (eBioscience) for 60 minutes. Cells were gated on the CD3⁺ population for analysis of Th1, Th2, or Th17 cells.
For detecting the proportion of CD4⁺ CD25⁺ Treg cells, intracellular Foxp3 staining was performed according to the manufacturer’s protocol of the Mouse Regulatory T Cell Staining Kit (eBioscience). Briefly, 2 × 10⁶ splenocytes, lymphocytes or liver cells from schistosome-infected or normal mice were surface-stained with anti-CD3-PerCP, anti-CD4-FITC and anti-CD25-APC for 30 minutes, followed by fixation and permeabilization with Cytofix/Cytoperm buffer (BD PharMingen) for 40 minutes and intracellular staining with anti-Foxp3-PE for 15 minutes. Cells were gated on the CD3⁺CD4⁺ population for analysis of Treg cells.

The spleens and lymph nodes were lightly ground between the frosted edges of two sterile glass slides, and the cell suspension was filtered through the 70-µm cell strainer. Immunosubsets from tumors, spleens, and lymph nodes were detected by flow cytometry (BD Biosciences, San Jose, CA). The frequency of CD8⁺IFN-γ⁺ T cells was assessed using intracellular cytokine staining. Purified cells were stimulated with PMA (10 ng/mL) and ionomycin (1 μg/mL) for 4 h and incubated for the last 1 h with brefeldin A (10 μg/mL). Cells were subjected to intracellular cytokine analysis using anti-IFN-γ antibody. The antibodies used for FACS included Pacific blue-conjugated anti-CD3, APC-conjugated anti-CD8, and PE-conjugated anti-IFN-γ (eBioscience). The data were analyzed using FlowJo software (TreeStar Inc., San Carlos, CA).