Discovery of an unexpected function of blood immunecells: Their ability to proliferate natimmunol
mice were injected i.p. with 1 mg EdU in 200 µl PBS 4 h before killing, unless otherwise stated. For all experiments involving EdU incorporation, 1 µg of PerCP-Cy5.5-conjugated anti-mouse CD45 was i.v. injected 10 min before killing to distinguish blood circulating and tissue leukocytes . For Csf1r-blocking experiments, 250 µg of anti-mouse Csf1r-blocking antibody or isotype control was injected i.v. 6 and 28 h after DT injection.
For BM cells, femurs and tibias were dissected and cleaned of soft adhering tissue. Distal and proximal ends were opened, and BM cells were flushed out. After centrifugation, cell pellets were resuspended in ice-cold PBS containing 10 mM EDTA and cell suspensions were filtered using a cell strainer to obtain a single-cell suspension.
Lungs, brains, liver and spleen were cut into small pieces with razor blades, and digested for 1 h at 37 °C in HBSS containing 5% vol/vol FBS , 1 mg mlDNase I . After 45 min of digestion, the suspension was flushed using a 18-gauge needle to dissociate aggregates. Ice-cold PBS containing 10 mM EDTA was added to stop the digestion process and cell suspensions were filtered using a cell strainer .
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