American Society of Hematology

Figure 5.

$Figure 5. TSP-1 scarcely binds to late apoptotic monocytes but extensively binds to iDCs. Viable and apoptotic monocytes and iDCs were washed twice with RPMI and incubated for 30 minutes on ice with 10 μg/mL TSP-1. Cells were then rewashed and stained with anti-TSP-1 antibody (Biomeda) followed by fluorescence-labeled secondary antibody, and with either FITC-labeled annexin V or propidium iodide (PI). (A) TSP-1 scarcely binds apoptotic monocytes. Double staining with anti-TSP-1-PE and annexin V-FITC is shown. Only 28% (18/64) of annexin V-FITC-positive monocytes bound TSP-1. Numbers indicate percentage of cells included in the respective quadrants. (B) TSP-1 binds late, rather than early, to apoptotic monocytes. Double staining with anti-TSP-1-FITC and PI is shown. Almost all monocytes that bind anti-TSP-1 are PI-positive late apoptotic cells. This is true also for the small fraction of PI-positive cells that is included in viable cells. (C) TSP-1 binds extensively to iDCs. TSP-1-bound iDCs had a mean fluorescence of 26, whereas viable and apoptotic monocytes show mean fluorescence of 9.14 and 9.73, respectively (P < .001). Data shown are representative of 3 experiments. Isotype control is shown as a gray-filled curve. Less than 5% of iDCs were annexin V positive and less than 1% were PI positive, excluding binding to iDCs due to apoptosis.$

TSP-1 scarcely binds to late apoptotic monocytes but extensively binds to iDCs. Viable and apoptotic monocytes and iDCs were washed twice with RPMI and incubated for 30 minutes on ice with 10 μg/mL TSP-1. Cells were then rewashed and stained with anti-TSP-1 antibody (Biomeda) followed by fluorescence-labeled secondary antibody, and with either FITC-labeled annexin V or propidium iodide (PI). (A) TSP-1 scarcely binds apoptotic monocytes. Double staining with anti-TSP-1-PE and annexin V-FITC is shown. Only 28% (18/64) of annexin V-FITC-positive monocytes bound TSP-1. Numbers indicate percentage of cells included in the respective quadrants. (B) TSP-1 binds late, rather than early, to apoptotic monocytes. Double staining with anti-TSP-1-FITC and PI is shown. Almost all monocytes that bind anti-TSP-1 are PI-positive late apoptotic cells. This is true also for the small fraction of PI-positive cells that is included in viable cells. (C) TSP-1 binds extensively to iDCs. TSP-1-bound iDCs had a mean fluorescence of 26, whereas viable and apoptotic monocytes show mean fluorescence of 9.14 and 9.73, respectively (P < .001). Data shown are representative of 3 experiments. Isotype control is shown as a gray-filled curve. Less than 5% of iDCs were annexin V positive and less than 1% were PI positive, excluding binding to iDCs due to apoptosis.

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