Anti cd24 pe

Stem/progenitor cell populations were identified and isolated as CD44⁺/CD24^-/low by FACS (Beckman Coulter MoFlo XDP sorter). The following antibodies and isotype controls were used: FITC-anti-CD44 (eBioscience 11-0441, 1:100 dilution), PE-anti-CD24 (eBioscience 12-0247, 1:20 dilution), APC-anti-CD44 (eBioscience 17-0441, 1:167 dilution), FITC-anti-CD24 (eBioscience 11-0247, 1:20 dilution), FITC-rat isotype control (eBioscience 11-4031, 1:100 dilution), PE-mouse isotype control (eBioscience 9012-4714, 1:20 dilution), APC-rat isotype control (eBioscience 17-4031, 1:167 dilution), FITC-mouse isotype control (eBioscience 11-4714, 1:20 dilution). Briefly, 5x10⁵ cells were incubated with antibodies in the dark at 4 °C for 1 h. Cells were washed and re-suspended in complete medium for analysis. For cell sorting, 10⁶ cells in 100 μL of complete medium were stained as described above. Cells were then treated with DNase I (Qiagen) in RDD buffer (10 mM Tris-HCL, 2.5 mM MgCl2, 0.5 mM CaCl2, pH7.6) for at least 15 min at room temperature, and passed through a 40 μm mesh (Becton Dickinson LTD) to avoid cell clumping. Data were analysed with the Weasel software.

The frequencies of immune cells including Th1, Th2, Th17, regulatory T cell (Treg), Tfh, and Breg in the peripheral blood were measured by flow cytometry. Briefly, PBMCs were isolated and incubated with PMA (10 ng/ml, eBioscience) and BFA (10 μg/ml, eBioscience) for 4 h then harvested and washed twice for 30 min. Then, cells were stained with anti-CD4 and anti-CD25 for 30 min at 4°C. During the intracellular staining, antibodies against IFN-γ, IL-4, and IL-17A were according to stain Th1, Th2, and Th17, respectively. Intracellular FoxP3 was also stained, and CD4⁺FoxP3⁺ was used to determine Treg. CD19⁺CD24^HighCD38^High cells were determined as Breg. CD4⁺PD1⁺CXCR5⁺ cells were determined as Tfh. The following anti-human antibodies for surface staining or intracellular staining were used: PE-CY7-anti-CD4, PE-CY5.5-anti-CD25, FITC-anti-IL-17A, PE-anti-Foxp3, FITC-anti-IFN-γ, PE-anti-IL-4, FITC-anti-CD19, PE-anti-CD24, PE-CY7-anti-CD38, PE-anti-PD1, and FITC-anti-CXCR5 (all eBioscience).

Cells exposed to indicated concentration of flubendazole for 72 hr, were trypsinized, washed twice with PBS by centrifugation (1,000 rpm, 5 min), and blocked (0.5% BSA/PBS) for 10 min. Collected cells were then incubated with anti-CD24 PE (eBioscience) and anti-CD44 FITC (eBioscience) or isotype controls antibodies according to manufacturer's instructions for 30 min at 4 °C protected from light. Then, cells were suspended in 1 ml PBS and subjected to flow cytometry analysis (Beckman). Side-scatter and forward-scatter profiles were used to eliminate cell doublets [51 (link)]. The statistical results of three independent experiments were presented.