Fig 1: Representative results of adiponectin-expressing Treg harvesting. Lymphocytes were obtained from a culture of cells from a previously developed murine model of a human micronodular thymic tumor with lymphoid stroma. 16 , 17 Control nonstained cells are shown in (a). Lymphoid cells were partially stained on the cell surface with anti-CD4 and anti-CD25 antibodies (b). CD4- and CD25-positive lymphoid cells were sorted using an SH800S cell sorter (c). Subsequently, the cells were immunocytostained with antibodies. Nonimmunostained cells (stained with 4',6-diamidino-2-phenylindole [DAPI]) are shown in (d). Sorted cells exhibited FOXP3 and adiponectin immunoreactivity. In (e), the green signal indicates adiponectin immunoreactivity and the pink color indicates the merging of red FOXP3 immunoreactivity and blue DAPI staining. Scale bar, 20 µm. The immunoblotting results indicated that the sorted cells expressed high molecular weight (HMW) adiponectin, as indicated by the arrow (f; upper column). The glyceraldehyde-3-phosphate dehydrogenase (G3PDH) band is shown (f; lower column).
Fig 2: Representative immunostaining of lymphoid stroma. (a) Immunofluorescent cytostaining. Lymphoid cells partially exhibited staining on the cell surface with anti-CD4 and CD25. Rat anti-mouse CD4 (clone GK1.5) conjugated with phycoerythrin (PE) (catalog no. 1102040: Sony), while rabbit monoclonal antibody to mouse CD25 conjugated with FITC (catalog no. 50292-M08H: Sino Biological Inc). In brief, cells were incubated with or without 1:100 diluted antibodies at 4°C for 1 h. After washing with phosphate-buffered saline (PBS), the cells were analyzed with a Guava EasyCyte cell analyzer (Hayward). (b) Double immunohistochemical staining. Note the cytoplasmic adiponectin (blue staining) and nuclear FOXP3 immunoreactivity (brown staining) in murine model tissue sections. The arrow indicates the adiponectin positive but FOX3P- lymphocyte, and the arrowhead indicates the adiponectin expressing FOXP3+ Tregs. For the detection of adiponectin and FOXP3, the tissues were formalin fixed and paraffin embedded, cut in section, incubated with anti-adiponectin and FOXP3 antibodies, and stained using MACH 2 Double Satin 2 kit (Biocare Medical) as previously described. 6 We used rabbit anti-adiponectin antibody (cat. no. GTX107737, GeneTex Inc.) and mouse anti-FOXP3 antibody (clone 3G3, Proteintech). Note that both antibodies are known to react with murine FOXP3.
Fig 3: Characterization of cultured T cell fraction composed of adiponectin-expressing Tregs, A-TregTF. Upper panel: Control nonstained cells are indicated as mock cells. Representative cell surface staining of A-TregTF with anti-CD4 and anti-CD25 antibodies (A-TregTF). Lower panel: Cells were subjected to immunocytostaining with specific antibodies. A-TregTF exhibits immunoreactivity to both FOXP3 and adiponectin. The green signal indicates adiponectin immunoreactivity and magenta represents the merging of red FOXP3 immunoreactivity and blue 4', 6-diamidino-2-phenylindole (DAPI) staining highlighting the cell nuclei. Scale bar: 20 µm.
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