Isolation and Differentiation of Human Monocytes

Human monocytes were isolated from healthy blood donors as previously described, using the RosetteSep monocyte-enrichment kit (StemCell) [23 (link)]. Following this negative separation procedure, over 85% of isolated monocytes were found to be CD14-positive [26 (link)]. For monocyte-macrophage differentiation, 1.2x10⁶ cells/9.6 cm² were cultured in RPMI1640 medium (with GlutaMax) (Invitrogen, Merelbeke, Belgium) supplemented with 10% FBS (Lonza, Basel, Switzerland), 100 U/mL penicillin and 100 μg/mL streptomycin (Invitrogen), in the presence of 50 ng/mL of macrophage colony-stimulating factor (M-CSF) (ImmunoTools, Friesoythe, Germany). After 7 days, cell culture medium was replaced without M-CSF renewal.

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Pinto A.T., Pinto M.L., Velho S., Pinto M.T., Cardoso A.P., Figueira R., Monteiro A., Marques M., Seruca R., Barbosa M.A., Mareel M., Oliveira M.J, & Rocha S. (2016). Intricate Macrophage-Colorectal Cancer Cell Communication in Response to Radiation. PLoS ONE, 11(8), e0160891.

Publication 2016

RosetteSep monocyte-enrichment kit GlutaMax FBS penicillin streptomycin M-CSF)

Blood donors Cell Cell culture Culture medium Human Macrophage Macrophage colony stimulating factor Monocyte Penicillin Stemcell Streptomycin

Corresponding Organization : University of Dundee

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Variable analysis

independent variables

Isolation of human monocytes from healthy blood donors using the RosetteSep monocyte-enrichment kit
Monocyte-macrophage differentiation by culturing 1.2x10^6 cells/9.6 cm^2 in RPMI1640 medium supplemented with 10% FBS, 100 U/mL penicillin, 100 μg/mL streptomycin, and 50 ng/mL of macrophage colony-stimulating factor (M-CSF)
Removal of M-CSF from the cell culture medium after 7 days

dependent variables

Percentage of CD14-positive monocytes after the isolation procedure
Monocyte-macrophage differentiation as a result of the culture conditions

control variables

Cell culture medium (RPMI1640 with GlutaMax, 10% FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin)
Cell seeding density (1.2x10^6 cells/9.6 cm^2)
Duration of the monocyte-macrophage differentiation (7 days)

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

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