Figure 2
Figure 2. PECAM-1−/− MKs demonstrate normal in vitro nuclear and cytoplasmic maturation, normal production of functional platelets, and proplatelet formation. (A) Flow-cytometric analysis of MKs. (i) Propidium iodide (PI) fluorescence histogram showing ploidy distribution of MKs (ploidy subclasses are indicated). (ii-iii) Fluorescence histograms of cells stained with FITC-CD41 (ii) or FITC-CXCR4 (iii). Gray line indicates irrelevant antibody control; black line, FITC-CD41 or FITC-CXCR4 antibodies. au indicates arbitrary unit. (B) Normal functional platelet number produced by PECAM-1−/− MKs. In order to isolate the platelet-like particles produced, the whole-cell population from the lower chamber was centrifugated at 1000g in the presence of PGI2 to avoid platelet preactivation. The pellet including transmigrated MKs, proplatelet-forming MKs, some HUVECs, cell debris, and platelets was then resuspended in Tyrode buffer and stimulated with or without thrombin to identify and isolate the functional platelet-like particles. Platelets generated in response to SDF1α and HUVECs from PECAM-1−/− MKs are functional and express P-selectin after thrombin stimulation, as shown by the representative fluorescence-activated cell sorter (FACS) histogram (i; gray line indicates unstimulated platelets; black line, thrombin-stimulated platelets), with the functional platelet counts represented as the mean of 3 experiments ± SEM (ii). (C) Representative CD41 immunofluorescent image of a PECAM-1−/− MKs adhered to a HUVEC monolayer generating proplatelets (PPs). Cumulative analysis shows similar numbers of PECAM-1+/+ MKs and PECAM-1−/− MKs demonstrating proplatelet formation (n = 3). Scale bar = 20 μm. Error bars indicate standard error. Fluorescence images were obtained using a Leica DMIRE 2 inverted microscope (Leica, Milton Keynes, United Kingdom) equipped with a 40×/1.3 NA Plan-Apochromat objective lens and a Coolsnap Photometrics camera (Photometrics, Huntington Beach, CA). Slidebook 4.0 software (Intelligent Imaging Innovations, Denver, CO) and ImageJ software (National Institutes of Health, Bethesda, MD) were used to acquire and process images.

PECAM-1−/− MKs demonstrate normal in vitro nuclear and cytoplasmic maturation, normal production of functional platelets, and proplatelet formation. (A) Flow-cytometric analysis of MKs. (i) Propidium iodide (PI) fluorescence histogram showing ploidy distribution of MKs (ploidy subclasses are indicated). (ii-iii) Fluorescence histograms of cells stained with FITC-CD41 (ii) or FITC-CXCR4 (iii). Gray line indicates irrelevant antibody control; black line, FITC-CD41 or FITC-CXCR4 antibodies. au indicates arbitrary unit. (B) Normal functional platelet number produced by PECAM-1−/− MKs. In order to isolate the platelet-like particles produced, the whole-cell population from the lower chamber was centrifugated at 1000g in the presence of PGI2 to avoid platelet preactivation. The pellet including transmigrated MKs, proplatelet-forming MKs, some HUVECs, cell debris, and platelets was then resuspended in Tyrode buffer and stimulated with or without thrombin to identify and isolate the functional platelet-like particles. Platelets generated in response to SDF1α and HUVECs from PECAM-1−/− MKs are functional and express P-selectin after thrombin stimulation, as shown by the representative fluorescence-activated cell sorter (FACS) histogram (i; gray line indicates unstimulated platelets; black line, thrombin-stimulated platelets), with the functional platelet counts represented as the mean of 3 experiments ± SEM (ii). (C) Representative CD41 immunofluorescent image of a PECAM-1−/− MKs adhered to a HUVEC monolayer generating proplatelets (PPs). Cumulative analysis shows similar numbers of PECAM-1+/+ MKs and PECAM-1−/− MKs demonstrating proplatelet formation (n = 3). Scale bar = 20 μm. Error bars indicate standard error. Fluorescence images were obtained using a Leica DMIRE 2 inverted microscope (Leica, Milton Keynes, United Kingdom) equipped with a 40×/1.3 NA Plan-Apochromat objective lens and a Coolsnap Photometrics camera (Photometrics, Huntington Beach, CA). Slidebook 4.0 software (Intelligent Imaging Innovations, Denver, CO) and ImageJ software (National Institutes of Health, Bethesda, MD) were used to acquire and process images.

Close Modal
This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal