Migration of monocytes toward RPS or APS was performed with a modified Boyden chamber as described previously56 (link). In short, isolated monocytes were loaded onto the upper chamber. Control medium, resting (RPS), activated (APS), or MCP-1 was loaded in the lower chamber. After incubation for 4 h at 37 °C, the membrane was fixed with 100% ethanol and stained with May-Gruenwald/Giemsa. The membrane was mounted on glass slides, and five randomly selected images were taken (Nikon Eclipse Ni-U, ×20 objective). The number of migrated cells was counted for each well in several microscopic fields per condition using ImageJ software to quantify the cell count (ImageJ, National Institutes of Health, USA)57 (link).
Rohlfing A.K., Kolb K., Sigle M., Ziegler M., Bild A., Münzer P., Sudmann J., Dicenta V., Harm T., Manke M.C., Geue S., Kremser M., Chatterjee M., Liang C., von Eysmondt H., Dandekar T., Heinzmann D., Günter M., von Ungern-Sternberg S., Büttcher M., Castor T., Mencl S., Langhauser F., Sies K., Ashour D., Beker M.C., Lämmerhofer M., Autenrieth S.E., Schäffer T.E., Laufer S., Szklanna P., Maguire P., Heikenwalder M., Müller K.A., Hermann D.M., Kilic E., Stumm R., Ramos G., Kleinschnitz C., Borst O., Langer H.F., Rath D, & Gawaz M. (2022). ACKR3 regulates platelet activation and ischemia-reperfusion tissue injury. Nature Communications, 13, 1823.
Other organizations :
University of Tübingen, University of Würzburg, German Cancer Research Center, Essen University Hospital, Universitätsklinikum Würzburg, Istanbul Medipol University, University College Dublin, Jena University Hospital, University of Lübeck
Positive control: MCP-1 (known chemoattractant for monocytes)
Negative control: Control medium
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