Blood samples were obtained from healthy subjects and patients with MPNs. CD45+ hematopoietic progenitor cells from peripheral blood were separated by immunomagnetic bead selection (Miltenyi Biotec, Bologna, Italy), as previously described.29 (link) Cells were cultured for 14 days, in Stem Span medium (STEMCELL Technologies Inc, Vancouver, BC, Canada) supplemented with 10 ng/ml TPO, IL-6 and IL-11 at 37 °C in a 5% CO2 fully humidified atmosphere.
Immunomagnetic bead selection
Manufactured by Miltenyi Biotec
Sourced in Italy
Immunomagnetic bead selection is a laboratory technique that utilizes magnetic beads coated with specific antibodies or ligands to isolate and purify target cells or molecules from complex biological samples. The core function of this technology is to selectively bind and separate the desired cell population or analyte for further analysis or downstream applications.
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4 protocols using immunomagnetic bead selection
1
Culturing Hematopoietic Progenitor Cells
2
Isolation and Differentiation of Human Macrophages
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Human peripheral blood mononuclear cells were isolated by Ficoll separation from the blood of healthy volunteers. CD14+ cells were positively selected using immunomagnetic bead selection (Miltenyi Biotec, Auburn, CA). Macrophage differentiation was performed over 5 days by culturing monocytes (5 × 105 cells/mL) in RPMI supplemented with 10% FBS, M-CSF (25 ng/mL), penicillin/streptomycin, and glutamine. MDMs were polarized using the protocol described above.
3
Expansion of Human Cord Blood CD34+ Progenitors
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CD34+ progenitors were obtained by immunomagnetic bead selection (Miltenyi Biotec, Bergisch-Gladbach, Germany) from human umbilical cord blood units collected according to the guidelines of the Ethics Committee of RWTH Aachen University (EK187/08). CD34+ progenitors were cultured in StemSpan serum-free medium in the presence of SCF (50 ng), TPO (20 ng), Flt3-L (50 ng), and IL-6 (10 ng, all Peprotech, London, UK) as before [7 (link), 8 (link)].
4
Cord Blood CD34+ Cell Differentiation
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Human cord blood was collected from the local blood bank following normal pregnancies and deliveries with informed consent of the parents, in accordance with the ethical committee of the IRCCS Policlinico San Matteo Foundation and the principles of the Declaration of Helsinki. CD34 + cells from cord blood samples were separated by immunomagnetic bead selection (Miltenyi Biotec, Bologna, Italy) and differentiated, as previously described ( 16), in Stem Span medium supplemented with 10 ng/ml thrombopoietin, IL-6 and IL-11 at 37 °C in a 5 % CO 2 fully humidified atmosphere.
Pro-platelet yields were evaluated at the end of the culture (13 days), as previously described (16) . Briefly, cells were seeded in a 24-well plate and incubated at 37 °C in a 5 % CO 2 fully humidified atmosphere. After 16 hours (h), pro-platelet-bearing megakaryocytes were counted by phase-contrast microscopy (Nikon TMS-F, Tokyo, Japan). Pro-platelets were identified as cells displaying long filamentous structure, ending with tips of the size of a platelet and their number was expressed as percentage of total cell count. Before being seeded, cells were incubated in Stem Span medium containing 10 µM test compound (ticagrelor, TAM, TIM, MRS2211) or vehicle, dimethyl sulfoxide (DMSO) for 16 h.
Pro-platelet yields were evaluated at the end of the culture (13 days), as previously described (16) . Briefly, cells were seeded in a 24-well plate and incubated at 37 °C in a 5 % CO 2 fully humidified atmosphere. After 16 hours (h), pro-platelet-bearing megakaryocytes were counted by phase-contrast microscopy (Nikon TMS-F, Tokyo, Japan). Pro-platelets were identified as cells displaying long filamentous structure, ending with tips of the size of a platelet and their number was expressed as percentage of total cell count. Before being seeded, cells were incubated in Stem Span medium containing 10 µM test compound (ticagrelor, TAM, TIM, MRS2211) or vehicle, dimethyl sulfoxide (DMSO) for 16 h.
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