Abstract
A main function of splenic red pulp macrophages is the degradation of damaged or aged erythrocytes. Here we show that these macrophages accumulate ferrimagnetic iron oxides that render them intrinsically superparamagnetic. Consequently, these cells routinely contaminate splenic cell isolates obtained with the use of MCS, a technique that has been widely used in immunological research for decades. These contaminations can profoundly alter experimental results. In mice deficient for the transcription factor SpiC, which lack red pulp macrophages, liver Kupffer cells take over the task of erythrocyte degradation and become superparamagnetic. We describe a simple additional magnetic separation step that avoids this problem and substantially improves purity of magnetic cell isolates from the spleen.
Highlights
In this study, we describe that Magnetic cell separation (MCS) isolates from the spleen, and under certain conditions from the liver, are routinely affected by distinct macrophage contaminants and that these may profoundly affect experimental results
When we isolated CD11c+ dendritic cells (DCs) from this organ with the use of MCS, we noted that in addition to the two classical splenic DC subsets expressing either the marker CD8 or CD11b4,5, 20–30% of the cell isolate constituted a third subset (Fig. 1A, upper dot-plot). These cells showed high autofluorescence at various visible light frequencies and displayed the surface phenotype CD11clo F4/80+ MHC II+ CD8– CD11b– (Fig. 1B, Supplementary Figure 1), which is characteristic of red pulp macrophages (RPM)[6]
We here report that erythrocyte-degrading cells, especially RPM and in their absence Kupffer cells, are superparamagnetic and contaminate cell isolation by MCS
Summary
We describe that MCS isolates from the spleen, and under certain conditions from the liver, are routinely affected by distinct macrophage contaminants and that these may profoundly affect experimental results. This analysis confirmed strong superparamagnetic properties in RPM at room temperature, comparable to those in commercially available magnetic nanoparticles that served as a positive control, but not in splenocytes depleted of RPM or in lymph node cells lacking RPM (Fig. 2A).
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