Galectin 9 (GAL9) is a member of the β-galactoside–binding galectin family. Galectin-9 is found outside of cells and may be exported by non-classical pathways. Galectin 9 exhibits a variety of biological activities, the majority of which have focused on its immunomodulatory role toward lymphocytes, were it shows specific interactions with TIM-3, and can negatively regulate Th1 type immunity. It also can agglutinate red blood.In this case, we chose rabbit erythrocyte (RaE) to assay its ability of agglutination. A general procedure for hemagglutination assay (or haemagglutination assay; HA) is as follows, two-fold dilute the recombinant Rat GAL9 with 0.9% sodium chloride injection, add 50μL a serial dilution of GAL9 to each well of a U or V-bottom shaped 96-well microtiter plate. The final well serves as a negative control with no GAL9, replace with 50μL 0.9% sodium chloride injection. Then add 50μL 1% rabbit erythrocyte to each well and mixed gently. The plate is incubated for 3 hours at room temperature. The results are shown in Figure 1. It was obvious that the minimal effective concentration of GAL9 is 6.25 μg/mL.

Figure 2. The hemagglutination assay of GAL9 in V- bottom shaped 96-well microtiter plate.