Fig. 5.
hCD33 negatively regulates cargo uptake in human monocytic cell lines. Targeted deletion of CD33 by CRISPR-Cas9 in a U937 and b THP-1 cells as demonstrated by flow cytometry staining with αhCD33 (clone WM53). Seven different clones of U937 (c–f) or three different clones of THP-1 (g–j) were tested in flow cytometry-based uptake of c, g dextran particles, d, h polystyrene beads, e, i myelin, and f, j aggregated Aβ1-42. For U937 cells (c–f), a representative flow cytometry histogram is shown for each cargo and summary plot of each clone. k Representative images of phagocytosis of polystyrene beads by U937 cells visualized by imaging flow cytometry. l In three independent experiments, hCD33−/− U937 cells have a higher percentage of cells with internalized polystyrene beads. m Lentiviral expression of hCD33M in hCD33−/− cells demonstrating that hCD33 expression levels (red) are higher than those of the parent parental U937 cell line expressing endogenous hCD33 (black). Statistical significance based on an unpaired Student’s T-test. n Three independent stable cell lines transduced to express empty lentivirus (black) or hCD33M (red) were analysis for phagocytosis with polystyrene beads by flow cytometry. Statistical significance based on a paired Student’s T-test.