Anti mouse monoclonal antibodies

Renal-infiltrated immune cells were screened for various cell surface and intracellular markers with flow cytometry. Briefly, 1 × 10⁶ cells were incubated with anti-mouse CD45, F4/80, CD4, CD8, CD11c, CD11b, Ly6G, and monoclonal antibodies conjugated with fluorescein isothiocyanate (FITC), phycoerythrin (PE), peridinin chlorophyll protein (PerCP), or allophycocyanin (APC) (all from BD Biosciences, San Jose, CA, USA) following the manufacturer's instructions. Immune cells derived from the kidneys were concomitantly stained for the intracellular content of TNF-α, IL-10, IL-17, and forkhead box P3 (FoxP3) by using the fixation/permeabilization kit and anti-mouse monoclonal antibodies conjugated with fluorescein isothiocyanate (FITC), phycoerythrin (PE), peridinin chlorophyll protein (PerCP), and allophycocyanin (APC) (BD Bioscience). For intracellular cytokine staining, cells were stimulated with 50 ng/mL PMA and 500 ng/mL ionomycin for 5 h, and GolgiStop (BD Biosciences) was added. Cells were fixed in Cytofix/Cytoperm, permeated with 0.1% saponin, and stained with fluorescent Abs. Flow cytometric analysis was conducted on a BD Biosciences FACSCalibur and analyzed by using the Flowing Software analysis program.

Lung-infiltrated immune cells were screened for various cell surface and intracellular markers with flow cytometry. Briefly, 1 × 10⁶ cells were incubated with anti-mouse CD45, F4/80, CD4, CD8, CD11c, CD11b, CD49b, FasL, CD107, perforin, NKG2D monoclonal antibodies conjugated with fluorescein isothiocyanate (FITC), phycoerythrin (PE), peridinin chlorophyll protein (PerCP), or allophycocyanin (APC) (all from BD Biosciences, San Jose, CA, USA) following the manufacturer's instructions. Immune cells derived from the lungs were concomitantly stained for the intracellular content of TNF-α, IL-10, and IL-17 by using the fixation/permeabilization kit and anti-mouse monoclonal antibodies conjugated with fluorescein isothiocyanate (FITC), phycoerythrin (PE), peridinin chlorophyll protein (PerCP), and allophycocyanin (APC) (BD Biosciences). For intracellular cytokine staining, cells were stimulated with 50 ng/mL PMA and 500 ng/mL ionomycin for 5 h, and GolgiStop (BD Biosciences) was added. Cells were fixed in Cytofix/Cytoperm, permeated with 0.1% saponin, and stained with fluorescent Abs. Flow cytometric analysis was conducted on a BD Biosciences' FACSCalibur and analyzed by using the flowing software analysis program.