Mouse anti penta his alexa fluor 647 conjugated monoclonal antibody

The reticulocyte-enriched samples were used for the flow cytometry-based direct-binding assay. Briefly, 1 × 10⁶ reticulocytes/ml or the same concentration of reticulocytes treated with each enzyme was incubated for 4 h at 25 °C with 0 to 20 μg of hexa-His-tagged recombinant PvRBP1a-34 or PvRBP1b-32 protein. PvDBP-RII and GST-His were used as the positive and negative controls for the reticulocyte binding assay, respectively. The samples were washed twice with 200 μl of PBS containing 1% BSA (PBS-BSA) and incubated with a mouse anti-penta-His Alexa Fluor 647-conjugated monoclonal antibody (Qiagen) for 1 h at 4 °C in the dark. The samples were washed three times with PBS-BSA and incubated with the Thiazole Orange (TO) Retic-COUNT reagent (Becton-Dickinson Co., San Jose, CA, USA) for 30 min at 25 °C. For the fluorescent detection of each RBC, a total of 100,000 events were acquired per sample using the FACS Accuri™ C6 Flow Cytometer (BD). The flow cytometric results were analyzed using FlowJo 7.6 (Treestar, Ashland, OR, USA). Unstained cells and cells singly stained with TO were used to separate the normocytes and reticulocytes, respectively.

The erythrocyte binding assay was performed as described previously (Tran et al., 2005 (link)). Briefly, a gradient concentration of purified PocDBP-RII protein was incubated with 1 × 10⁶/ml cells or the same concentration of reticulocytes treated with each enzyme for 3 hr at 25°C. The PvDBP-RII and GST-His protein reticulocyte binding activities were used as the experimental controls. The samples were washed with 200 µl of PBS (1% BSA) two times, followed by incubation with diluted (1:50) mouse anti-penta-His Alexa Fluor 647-conjugated monoclonal antibody (Qiagen, Hilden, Germany) for 1 hr at 4°C in the dark. The samples were washed three times with PBS (1% BSA) and incubated with TO (Becton-Dickinson Co., San Jose, CA) for 30 min at 25°C. A total of 100,000 events were counted per sample using a FACS Accuri™ C6 Flow Cytometer (Becton-Dickinson Co.). FlowJo 7.6 (Treestar, Ashland, OR) was used to analyze the flow cytometric results. Unstained cells and cells singly stained with TO represented normocytes and reticulocytes, respectively.