Anti-CD43 and anti-CD44 coating exert minor effects on cell behavior. (A) Freshly isolated murine HSCs (KSL CD150⁺CD48⁻CD34⁻CD135), MPP1 (KSL CD150⁺CD48⁻CD34⁺CD135⁻), MPP2 (KSL CD150⁺CD48⁺CD34⁺CD135⁻), MPP3 (KSL CD150⁻CD48⁺CD34⁺CD135⁻), MPP4 (KSL CD150⁻CD48⁺CD34⁺CD135⁺), MPP5 (KSL CD150⁻CD48⁻), pre-GMP (KL CD41⁻CD16/32⁻CD150⁻CD105⁺), GMP (KL CD41⁻CD16/32⁺CD150⁻), and pre-MegE (KL CD41⁻CD16/32⁻CD150⁺CD105⁻) were sorted and incubated in 100 ng/mL SCF, 100 ng/mL TPO, 10 ng/mL IL-3, and 2 U/mL EPO, 10% FCS on coated slides at indicated antibody or reagent concentration and imaged every hour. Cell death was measured continuously by cell segmentation and quantification of Annexin V “in culture” antibody staining for 60 hours. Quantification of cell survival (frequency of Annexin V⁻ cells) at 24 hours after culture start is displayed. Anti-CD43 and anti-CD44 coating had no or minor effects on cell survival across multiple HSPC populations. Triangles indicate increasing coating concentration range: 0, 10, and 20 μg/mL. n = 3 independent experiments, 83 to 252 cells per conditions, >2.7 × 10⁶ quantified data points across 600 time points; mean ± SEM. (B) Proliferation is reduced only on high anti-CD43 concentrations, but not on anti-CD44 coating. n = 3 independent experiments, 41 to 461 cells per condition, >20.5 × 10⁶ quantified data points (cells) across 160 measured time points total mean. SEM not shown for better readability. (C) Mixed, MegE, and myeloid colonies can be identified based on CD41 and CD16/32 “in culture” antibody staining. Scale bar, 100 μm. (D) Differentiation into myeloid (top) and MegE (bottom) lineage (measured by quantification of CD41 and CD16/32 “in culture” antibody staining after cell segmentation at day 6 of culture) is not affected by coating. n = 3 independent experiments, 41 to 461 cells per condition, >20.5 × 10⁶ quantified data points (cells) across 160 measured time points total. Triangles indicate increasing coating concentration range: 1, 5, and 20 μg/mL; mean ± SEM. (E) Human CD34⁺ CB cells were cultured in Iscove modified Dulbecco medium supplemented with 20% BIT, 100 ng/mL SCF, 100 ng/mL Flt3L, and 50 ng/mL TPO. Cell death was measured continuously by cell segmentation and quantification of Annexin V “in culture” antibody staining for 5 days. Effects on survival 24 hours after culture are displayed. Anti-CD43 and anti-CD44 coating do not affect survival. n = 4 independent experiments, 1144 to 1757 cells per condition, >3.9 × 10⁶ quantified data points (cells) across 320 measured time points total. Triangles indicate increasing coating concentrations: 0, 1, 5, and 20 μg/mL; mean ± SEM. (F) Proliferation of CD34⁺ CB (measured as number AnnexinV⁻ cells over time) for 5 days. Anti-CD43 and anti-CD44 do not affect proliferation. n = 4 independent experiments; 1951 to 3039 cells per condition, >18 × 10⁶ quantified data points (cells) across 320 measured time points total, mean. SEM not shown for better readability (G) CD34⁺ CB in vitro differentiation was measured by continuous quantification of CD34 “in culture” antibody staining for 5 days. Differentiation (measured by CD34 downregulation) is not affected by anti-CD43 or anti-CD44 coating. n = 2 independent experiments, 1358 to 2342 cells per condition, >16 × 10⁶ quantified data points (cells) across 320 measured time points total, mean, SEM not shown for better readability. Conc., concentration; n.a., not available; n.s., not significant.