Buffy coats from the venous blood of normal healthy volunteers were obtained by the Blood Transfusion Centre of Slovenia, in accordance with institutional guidelines. Peripheral blood mononuclear cells (PBMCs) were then isolated using Lymphoprep (Stemcell Technologies, Vancouver, Canada). The cells were washed twice with phosphatebuffered saline (PBS), counted, and used as a source for the immunomagnetic negative isolation of CD14-positive cells (EasySep, Stemcell Technologies, Vancouver, Canada). CD14-positive cells (the purity of CD14 + cells was always greater than 95%, as determined by flow cytometry) were cultured in an RPMI 1640 (Sigma-Aldrich, MO, USA) medium, supplemented with 10% fetal bovine serum (Gibco, NY, USA), 50 U/mL penicillin and 50 µg/mL streptomycin (Sigma-Aldrich, MO, USA), 800 U/mL of rhGM-CSF and 1000 U/mL of rhIL-4 (Biolegend, San Diego, CA) at 1 × 10 6 cells/mL. On day 3, half of the medium was exchanged with the addition of starting quantities of rhGMCSF and rhIL-4. After 5 days, non-adherent, immature DCs were harvested and characterized, by means of flow cytometry, as CD14neg, DC-SIGNhigh, CD80low, CD83high, CD86low, HLA-DRlow, CD85khigh, CD207alow.
Rhgm csf
Manufactured by BioLegend
Sourced in United States
RhGM-CSF is a recombinant human granulocyte-macrophage colony-stimulating factor. It is a cytokine that regulates the production, differentiation, and function of granulocytes and macrophages.
Automatically generated - may contain errors
Lab products found in correlation
3 protocols using rhgm csf
1
Isolation and Characterization of Immature Dendritic Cells
2
Stem Cell Sphere Formation Assay
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ALKBH5- and control RPMI8226 cells were plated in 6-well plates and cultured in prepared stem cell medium. Briefly, stem cell medium was prepared by RPMI 1640 medium supplemented with rh stem cell factor, rh GM-CSF, rh IL-3 and EPO (Biolegend, San Diego, CA, USA) and used immediately after preparation. The cell spheroids larger than 50μm in diameter under the microscope was counted as stem cell spheres. Sphere formation ability was determined by the number of stem cell spheroids that formed 14 days after seeding.
3
Deriving M1-like Macrophages from Donor Monocytes
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Due to the limited availability of pulmonary macrophages [19 (link)], donor monocytes were derived from normal healthy donor whole blood buffy coats using anti-human CD14 Magnetic Particles (BD ImagTM, Franklin Lakes, NJ #557769, USA) and cell separation magnets (BD ImagTM, Franklin Lakes, NJ #552311, USA). Purity was confirmed using flow cytometry as previously described [15 (link)]. Polarization was accomplished using 50 ng/mL rhGM-CSF (#572905, Biolegend, San Diego, CA, USA) for 7 days to obtain M1 (GM-MDMs, corresponds to GM-CSF-generated monocyte-derived macrophages [MDMs])-like macrophages, respectively [15 (link)].
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