American Society of Hematology

Figure 4.

$DPT alters the hematopoietic-endothelial cell interaction. (A) Adhesion to HUVECS. Cells were grown to confluency in a 96-well plate. Murine WBM cells were incubated with 2 µg/mL rDPT prior to transfer to endothelial-coated wells. Adhesion occurred over 2 hours, followed by washing and quantification of adhered cells by flow cytometry and counting beads. (B) Cell migration assay; 100 000 murine WBM cells were incubated in the presence of rDPT for 4 hours. The bottom chamber contained SDF1 (0, 1, 10, 50, or 100 ng/mL). The numbers of cells that migrated to the bottom chamber were enumerated by flow cytometry, normalized, and compiled. See supplemental Figure 5 for detailed data about each group. (C) Cell migration assay across endothelial cells; 500 000 SCA1+ cells were incubated with HUVEC-coated Transwells in the presence of rDPT for 4 hours. The numbers of cells that migrated to the bottom chamber were enumerated by flow cytometry. (D) Adhesion to HDMECs. HDMECs were grown to confluency in a 96-well plate and activated with 2 ng/mL TNF-α overnight. Murine WBM cells were labeled with CellTracker Green and incubated with rDPT prior to transfer to endothelial coated wells. Adhesion occurred over 1 hour followed by washing and quantification of adhered cells by fluorometry. (E) Release assay. HDMECs were grown to confluency in a 96-well plate and activated 2 ng/mL TNF-α overnight. Murine WBM cells were labeled with CellTracker Green and incubated with rDPT prior to transfer to HDMEC -containing wells. Adhesion occurred overnight, followed by gentle washing. Increasing amounts of rDPT were added in media for 1 hour at 37°C, followed by gentle washing. Quantification of adhered cells was done by fluorometry. (F) Release assay of lineage separated cells. Experiment was performed as in (E), with the exception that WBM cells underwent separation into lin+ and lin− fractions, prior to use, using magnetic bead isolation. rDPT was used at 5 µg/mL. Raw data for (D-F) are shown in supplemental Figure 6. (G) Schema for microfluidic device to assess HSPC-endothelial adherence under flow; 500 000 lin− cells per milliliter from a ubiquitously expressing EGFP+ mouse were flowed through an endothelialized lumen previously activated with TNF-α. Cell adherence was captured in real-time using fluorescent image capture. (H) Enumeration of cells that adhered to the endothelial wall during flow with and without the addition of 1 µg/mL rDPT (data are pooled from 2 independent experiments). Representative experiments (n = 3-6 wells per condition) of 2 or 3 biologic replicates are shown. All data are means and standard deviations, unless otherwise noted. *P < .05, Student t test. CTL, cytotoxic T lymphocyte; +/−, with or without.$

DPT alters the hematopoietic-endothelial cell interaction. (A) Adhesion to HUVECS. Cells were grown to confluency in a 96-well plate. Murine WBM cells were incubated with 2 µg/mL rDPT prior to transfer to endothelial-coated wells. Adhesion occurred over 2 hours, followed by washing and quantification of adhered cells by flow cytometry and counting beads. (B) Cell migration assay; 100 000 murine WBM cells were incubated in the presence of rDPT for 4 hours. The bottom chamber contained SDF1 (0, 1, 10, 50, or 100 ng/mL). The numbers of cells that migrated to the bottom chamber were enumerated by flow cytometry, normalized, and compiled. See supplemental Figure 5 for detailed data about each group. (C) Cell migration assay across endothelial cells; 500 000 SCA1⁺ cells were incubated with HUVEC-coated Transwells in the presence of rDPT for 4 hours. The numbers of cells that migrated to the bottom chamber were enumerated by flow cytometry. (D) Adhesion to HDMECs. HDMECs were grown to confluency in a 96-well plate and activated with 2 ng/mL TNF-α overnight. Murine WBM cells were labeled with CellTracker Green and incubated with rDPT prior to transfer to endothelial coated wells. Adhesion occurred over 1 hour followed by washing and quantification of adhered cells by fluorometry. (E) Release assay. HDMECs were grown to confluency in a 96-well plate and activated 2 ng/mL TNF-α overnight. Murine WBM cells were labeled with CellTracker Green and incubated with rDPT prior to transfer to HDMEC -containing wells. Adhesion occurred overnight, followed by gentle washing. Increasing amounts of rDPT were added in media for 1 hour at 37°C, followed by gentle washing. Quantification of adhered cells was done by fluorometry. (F) Release assay of lineage separated cells. Experiment was performed as in (E), with the exception that WBM cells underwent separation into lin⁺ and lin⁻ fractions, prior to use, using magnetic bead isolation. rDPT was used at 5 µg/mL. Raw data for (D-F) are shown in supplemental Figure 6. (G) Schema for microfluidic device to assess HSPC-endothelial adherence under flow; 500 000 lin⁻ cells per milliliter from a ubiquitously expressing EGFP⁺ mouse were flowed through an endothelialized lumen previously activated with TNF-α. Cell adherence was captured in real-time using fluorescent image capture. (H) Enumeration of cells that adhered to the endothelial wall during flow with and without the addition of 1 µg/mL rDPT (data are pooled from 2 independent experiments). Representative experiments (n = 3-6 wells per condition) of 2 or 3 biologic replicates are shown. All data are means and standard deviations, unless otherwise noted. *P < .05, Student t test. CTL, cytotoxic T lymphocyte; +/−, with or without.

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