Human neutrophil-derived MPO was either purified from buffy coats as described before53 (link), or obtained commercially (MY167 – EC1.11.1.7, Elastin Products Company, Inc.). Briefly, polymorphonuclear (PMN) cells were isolated from buffy coats by Ficoll density gradient centrifugation followed by dextran sedimentation. PMN cells were dissolved in cetyltrimethyl ammonium bromide (Sigma) and sonified to extract lysosomes. Further purification by concanavalin A-Sepharose and Sephadex G100 size exclusion chromatography was performed to capture the MPO fraction. The final MPO preparation had an OD 428/280 ratio of 0.76.
To stain MPO-reactive B cells, MPO was conjugated to Alexa Fluor 647 (AF647) by incubating MPO with 7 ug Alexa Fluor™ 647 NHS Ester (A375733, Invitrogen) per 100 mg MPO in 0.1 M NaHCO3 for 2 h at room temperature. Unbound label was removed with 30 kDa Amicon Ultra-0.5 Centrifugal Filter Units (Merck). Rate of labeling, as determined by ultraviolet-visible (UV-VIS) spectroscopy, was between 2.2 and 7.7. Conjugation of MPO to R-Phycoerythrin (PE) was performed using the LYNX Rapid RPE Antibody Conjugation Kit (LNK021RPE, Bio-Rad) according to the manufacturer’s instructions. As MPO was still enzymatically active and able to degrade coupled fluorophores, MPO inhibitor 4-Aminobenzoic hydrazide (ABAH, A41909-10G, Sigma Aldrich) was added at 100 mM both during conjugation reactions and storage, to avoid fluorochrome degradation.
To stain MPO-reactive B cells, MPO was conjugated to Alexa Fluor 647 (AF647) by incubating MPO with 7 ug Alexa Fluor™ 647 NHS Ester (A375733, Invitrogen) per 100 mg MPO in 0.1 M NaHCO3 for 2 h at room temperature. Unbound label was removed with 30 kDa Amicon Ultra-0.5 Centrifugal Filter Units (Merck). Rate of labeling, as determined by ultraviolet-visible (UV-VIS) spectroscopy, was between 2.2 and 7.7. Conjugation of MPO to R-Phycoerythrin (PE) was performed using the LYNX Rapid RPE Antibody Conjugation Kit (LNK021RPE, Bio-Rad) according to the manufacturer’s instructions. As MPO was still enzymatically active and able to degrade coupled fluorophores, MPO inhibitor 4-Aminobenzoic hydrazide (ABAH, A41909-10G, Sigma Aldrich) was added at 100 mM both during conjugation reactions and storage, to avoid fluorochrome degradation.
