Anti hil 9 percp cy5

Human peripheral blood T cells or murine T cells were fixed with cold methanol for 2 minutes, followed by permeabilization using 0.1% Triton X-100 for 3 hours. The fixed T cells were incubated with antibodies against DUSP8 (1:200), Pur-α (1:200, clone 1C10, Abcepta), BATF (1:200, 8638, Cell Signaling Technology), PU.1 (1:500, MA5-15064, Invitrogen), or IRF-4 (1:100, 4964, Cell Signaling Technology) at 4 °C for overnight, followed by incubation with Alexa Fluor-488 anti-rabbit IgG (1:400, ab150061, Abcam) and Alexa Fluor-594 anti-mouse IgG (1:400, ab150108, Abcam) antibodies for 1 hour. For detection of IL-9, the stained cells were further incubated with anti-hIL-9-PerCP-Cy5.5 (1:100, clone MH9A3, BD Biosciences) antibody for 2 hours. Cell nucleus was stained with DAPI. DAPI, 4′,6-diamidino-2-phenylindole. Confocal images were acquired by Leica TCS SP5 II confocal microscope.

Human PBLs were freshly isolated from 10 mL whole blood by centrifugation, followed by washing twice with 50 mL ACK (ammonium-chloridepotassium) buffer. 1 × 10⁶ PBLs were first incubated in MACS buffer containing anti-hCD3-PE-Cy7 (1:100, BD Biosciences) and GolgiStop (1:1,000, BD Biosciences) for 3 hours at room temperature without any other stimulation (32 (link)). The PBLs were incubated in 200 μL Cytofix/Cytoperm buffer (BD Biosciences) at 4 °C overnight. The permeabilized cells were washed with Perm/Wash buffer (BD Biosciences) and then incubated in the Perm/Wash buffer containing anti-DUSP8 antibody (1:500) and anti-hIL-9-PerCP-Cy5.5 (1:50) at 4 °C for 2 hours, followed by staining with anti-rabbit IgG-FITC (1:400) for 1 hour. After washing with MACS buffer, the cells were detected by FACSCanto II flow cytometer (BD Biosciences). Notably, when we processed clinical samples using a previously published method (34 (link)), the frequency of IL-9-producing T cells in an asthma patient shown in Figure 6B would be decreased from 10.2% to 1.17%. It is likely that the incubation time for GolgiStop and permeabilization is critical.