CD126 antibody | B-R6

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	Mononuclear cells (MNCs) were separated by Ficoll-Hypaque density gradient centrifugation. Immunostaining was performed by a standard indirect immunofluorescence method, as previously described

Protocol tips
Mononuclear cells (MNCs) were separated by Ficoll-Hypaque density gradient centrifugation. Immunostaining was performed by a standard indirect immunofluorescence method, as previously described

Publication protocol

"Mononuclear cells (MNCs) were separated by Ficoll-Hypaque density gradient centrifugation. Immunostaining was performed by a standard indirect immunofluorescence method, as previously described [7,12]. Monoclonal antibodies (mAbs) were used to detect the following cell surface markers and cytokine receptors: CD4, CD7, CD11c, CD18, CD19, CD34, and CD56 (Becton Dickinson, Mountain View, CA); CD2, CD11b, HLA-DQ, and HLA-DR (Ortho, Raritan, NJ); CD14 and CD33 (Coulter, Hialeah, FL); CD13 (CALTAG Laboratories, Burlingame, CA); IL-3Rα (N3A) and βc (5A5) (kindly provided by Dr. T. Kitamura, Tokyo University, Japan); IL-2Rα (anti-Tac) (Dr. T. Uchiyama, Kyoto University, Japan); IL-2Rβ (Mikβ1) (Dr. M. Tsudo, Osaka Red Cross Hospital, Japan); GM-CSFRα, IL-4Rα, IL-7Rα, and c-mpl (Genzyme, Cambridge, MA); c-kit, IL-6Rα, and GP130 (Serotec, Oxford, UK); IL-5Rα, G-CSFR, and γc (Pharmingen, San Diego, CA); c-fms (Santa Cruz Biotechnology, Santa Cruz, CA); and FLT3 (Immunotech, Marseille, France). Regarding anti-Tac, it was firstly developed mAb against IL-2Rα [20], and has been used to detect IL-2Rα in various hematologic malignancies in many studies [7,12,21,22].

A fluorescein isothiocyanate-conjugated goat anti-mouse IgG F(ab)2 (GAM-FITC; Coulter) was used as the secondary antibody. Whole mouse Igs, IgG1 and IgG2 (Chemicon, Temecula, CA) were used as the controls. Double immunostaining was performed to determine whether myeloid antigen and IL-2Rα were expressed on the same or different cells. Briefly, cells were first stained with the anti-Tac mAb with indirect fluorescence, and with a phycoerythrin (PE)-conjugated anti-CD13 antibody. Flow cytometric analysis was performed using a Cytron flow cytometer (Ortho). As the cell samples were found to consist of ≥90% leukemia cells in cytospin preparations, samples for the cell surface markers were considered positive if more than 15% of the leukemia cells showed immunofluorescence that was greater than that observed with the negative control. Expression levels of individual cytokine receptors were assessed by measuring the mean fluorescence intensities (MFIs) of cell stained with the related antibodies. Antibody binding capacities (sites/cell) of test samples and control cells were calculated based on MFIs and calibration curves obtained using the DAKO QIFIKIT and TallyCAL software packages (DAKO, Grostrip, Denmark), as described previously [12]. Samples with <200 binding sites/cell were judged as undetectable in this study."

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