The BM of MPN patients were collected and processed for single-cell preparation for flow cytometry and enrichment of CD163+ monocytes/macrophages. The single cell suspensions were prepared as previously described [18 (link), 29 (link)]. Then, BM CD163+ monocytes/macrophages from HCs and MPN patients were enriched with the CD163 MicroBead Kit (Miltenyi, Cat#:130–124-420), according to the manufacturer's instructions. BM cells were first incubated with CD163-biotin for 15 min on ice in the dark. The cells were then washed with PBS plus 2% FBS and 2 mM EDTA, centrifuged at 1200 rpm for 10 min, and then incubated with anti-biotin microbeads for 15 min on ice in the dark. The cells were again washed with PBS plus 2% FBS and 2 mM EDTA, again centrifuged at 300 g for 10 min, and then resuspended with 2-mL PBS plus 2% FBS and 2 mM EDTA. Finally, the BM CD163+ monocytes/macrophages were enriched with Quadro MACS, according to the manufacturer’s instructions.
Cd163 microbead kit
Manufactured by Miltenyi Biotec
Sourced in United States
The CD163 MicroBead Kit is a product designed for the isolation and enrichment of CD163-positive cells from various sample types. It contains magnetic beads coated with antibodies specific to the CD163 surface marker, enabling the targeted separation of cells expressing this receptor.
Automatically generated - may contain errors
2 protocols using cd163 microbead kit
1
Enrichment of BM CD163+ Monocytes/Macrophages
2
Isolation and Culture of Primary Hepatic Cells
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After a two-step collagenase perfusion, the liver extract was filtered and centrifuged, as previously described. 18 (link) PHH were cultured on a collagen layer and maintained in Williams E medium (Gibco, Billings, MT, USA) supplemented with 5% FCII serum (Cytiva, Marlborough, MA, USA), 50 U/mL of penicillin/streptomycin, 5 µg/mL of bovine insulin, 2% DMSO (Sigma-Aldrich, St. Louis, MO, USA), 1x Glutamax (Gibco) and 5×10 -5 M of hydrocortisone (SERB, Brussels, BE). KCs were purified from the non-parenchymal cell mixture by a two-phase iodixanol gradient (Optiprep, BioVision, Waltham, MA, USA), followed by positive selection with the CD163 MicroBead kit (Miltenyi Biotec, Bergisch Gladbach, DE). KCs were seeded at 3×10 5 cells/well into 24-well plates and cultured in RPMI-1640 medium (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% FCII serum and 50 U/mL of penicillin/streptomycin. HepaRG cells were cultured and DMSO-differentiated as previously described. 19 (link) All cells were cultured at 37°C in a humidified 5% CO 2 incubator.
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