Figure 1.
Anti-CD43 and anti-CD44 coatings reduce HSPC motility and enable medium exchange without cell-identification loss. (A) Freshly isolated KSL were sorted and incubated in 100 ng/mL stem cell factor (SCF), 100 ng/mL thrombopoietin (TPO), 10 ng/mL interleukin-3 (IL-3), 2 U/mL erythropoietin (EPO), and 10% fetal calf serum (FCS) on coated slides at indicated antibody or reagent concentrations and imaged every 2.5 minutes. (B) Representative images of KSL and human CD34+ CB on coated slides after 3 days. Colony formation (arrowheads) can only be recognized due to cell immobilization on anti-CD43 and anti-CD44 coating. In other conditions, forming colonies scatter and mix due to cell displacement. Scale bar, 100 μm. (C) Representative video frames of fixed region of interest (ROI) with increasing time intervals. Cells on uncoated slides are highly motile preventing reliable assignment of cell identifications (movement >1 cell diameter) over time and leave the ROI after about 40 minutes. Triangles indicate increasing coating concentration range: 1, 5, and 20 μg/mL. Scale bar, 50 μm. (D) Quantification of cell motility in micrometers per 2.5 minutes. Anti-CD43 reduces cell motility stronger and at lower concentration than anti-CD44 coating. n = 1 with 42-73 cells and 2124 to 4455 time points per condition analyzed. (E) Frequency of cell movements >1 cell diameter; 5 μg/mL anti-CD43–coating concentration is sufficient to keep single-cell identifications with high confidence. Triangles indicate increasing coating concentration range: 1, 5, and 20 μg/mL. (F-G) Cell and cell-identification loss (movement >1 cell diameter between 2 measurements) after medium exchanges. Virtually no or minor cell losses on slides coated with 20 μg/mL anti-CD43 and anti-CD44. Fibronectin, retronectin, and poly-l-lysine were used at 50 μg/mL, 100 μg/mL, 0.01% (wt/vol), respectively. n = 3 independent experiments, 260 to 298 and >62 to 306 cells per condition, respectively; mean ± SEM; χ2 test. FN, fibronectin; IgM, immunoglobulin M; PLL, poly-l-lysine; RN, retronectin; Unc., uncoated.

Anti-CD43 and anti-CD44 coatings reduce HSPC motility and enable medium exchange without cell-identification loss. (A) Freshly isolated KSL were sorted and incubated in 100 ng/mL stem cell factor (SCF), 100 ng/mL thrombopoietin (TPO), 10 ng/mL interleukin-3 (IL-3), 2 U/mL erythropoietin (EPO), and 10% fetal calf serum (FCS) on coated slides at indicated antibody or reagent concentrations and imaged every 2.5 minutes. (B) Representative images of KSL and human CD34+ CB on coated slides after 3 days. Colony formation (arrowheads) can only be recognized due to cell immobilization on anti-CD43 and anti-CD44 coating. In other conditions, forming colonies scatter and mix due to cell displacement. Scale bar, 100 μm. (C) Representative video frames of fixed region of interest (ROI) with increasing time intervals. Cells on uncoated slides are highly motile preventing reliable assignment of cell identifications (movement >1 cell diameter) over time and leave the ROI after about 40 minutes. Triangles indicate increasing coating concentration range: 1, 5, and 20 μg/mL. Scale bar, 50 μm. (D) Quantification of cell motility in micrometers per 2.5 minutes. Anti-CD43 reduces cell motility stronger and at lower concentration than anti-CD44 coating. n = 1 with 42-73 cells and 2124 to 4455 time points per condition analyzed. (E) Frequency of cell movements >1 cell diameter; 5 μg/mL anti-CD43–coating concentration is sufficient to keep single-cell identifications with high confidence. Triangles indicate increasing coating concentration range: 1, 5, and 20 μg/mL. (F-G) Cell and cell-identification loss (movement >1 cell diameter between 2 measurements) after medium exchanges. Virtually no or minor cell losses on slides coated with 20 μg/mL anti-CD43 and anti-CD44. Fibronectin, retronectin, and poly-l-lysine were used at 50 μg/mL, 100 μg/mL, 0.01% (wt/vol), respectively. n = 3 independent experiments, 260 to 298 and >62 to 306 cells per condition, respectively; mean ± SEM; χ2 test. FN, fibronectin; IgM, immunoglobulin M; PLL, poly-l-lysine; RN, retronectin; Unc., uncoated.

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