Concentration columns

Inhibition of the CSF1R on CD14+ human monocytes was accomplished with a neutralizing antibody targeting the CSF1R (R&D Systems clone 61701). For neutralization of the CSF1R in mouse PyMT breast tumors, 50 mg/kg body weight of ASF98 rat anti-mouse monoclonal c-fms antibody was used. Dr. Shin-Ichi Nishikawa kindly provided the ASF98 hybridoma (Riken Center for Developmental Biology, Kobe, Japan). Antibodies were produced and isolated as previously described [30] (link), [31] (link). Briefly, hybridoma cells were grown in serum-free media (Gibco) until the media exhausted. Cell-free supernatant was collected by centrifugation and loaded onto a protein A/G agarose bead column. Antibody was then eluted using elution buffer (Thermo Scientific). Elution fractions were neutralized with 1.5 M Tris base (pH 8.0) and pooled fractions were concentrated by centrifugation through concentration columns (Millipore). Antibody concentration was determined using the Bradford colorimetric assay and samples compared to BSA protein standards.

Lipoprotein isolation was performed using the protocol described previously^{60 (link)}. In brief, lipoproteins from mouse serum and ChP primary culture were separated from soluble proteins using KBr discontinuous gradient. To remove excessive KBr, fractions were filtered using concentration columns (Millipore) and washed several times in PBS. Finally, the ultrafiltrate of lipoproteins fractions was resuspended in PBS before further applications. Used reagents are listed in the Supplementary Table 1.