Anti cd14 antibody conjugated magnetic microbeads

The study protocol was approved by the Ethics Committee of Showa University School of Medicine (Tokyo, Japan; approval number: 2799). Written informed consent was obtained from all T1D patients and healthy volunteers. The study was designed in compliance with the Declaration of Helsinki.
Five patients with uncontrolled T1D despite multiple daily insulin injections over ≥12 weeks and six controls were enrolled in the present study. Blood samples were collected, and human monocyte-derived macrophages were isolated using anti-CD14 antibody-conjugated magnetic microbeads (Miltenyi Biotec, Auburn, CA, USA) as previously described [14 (link)].

To generate monocyte-derived DCs, human peripheral blood mononuclear cells (PBMC), obtained under informed consent from healthy donors, were isolated by Fycoll-Paque gradient (Pharmacia). CD14 + monocytes were positively selected using anti-CD14 antibody-conjugated magnetic microbeads (Miltenyi Biotec). To generate immature DCs, purified monocytes were then cultured in 12-well plates for 6 days, at a density of 10⁶ cells/3 mL in RPMI 1640 containing 10% FCS, 2 mM L-glutamine, 100 U/mL penicillin G, 100 mg/mL streptomycin, 50 ng/mL recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF) and 20 ng/mL interleukin-4 (IL-4) (Miltenyi Biotec). Cytokines were replenished every other day, along with 20% fresh medium. SW480 cells grown on coverslips treated or mock treated with βGBP for 48 h were washed three times in PBS to remove βGBP, co-incubated with DC’s for 4 h at 4 °C (1/3 ratio) and finally washed in PBS to remove unbound DCs. The cells were fixed with 4% paraformaldehyde in PBS for 30 min at 25 °C, stained for CRT (Affinity Bioreagents) or the CD1a DC marker (Miltenyi Biotec) and observed as above.