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Fig. 1 | Arthritis Research & Therapy

Fig. 1

From: Native myeloperoxidase is required to make the experimental vasculitis model

Fig. 1

Development of MPO-AAV. a ANCA detected by FCM. Human peripheral blood neutrophils were fixed with 4% paraformaldehyde, and then the plasma membrane of neutrophils was penetrated using permeabilization wash buffer (BioLegend, San Diego, CA, USA). Cells (1 × 106/ml) were allowed to react with 1:500 diluted rat sera for 30 min at room temperature (RT) followed by reaction with fluorescence-labeled secondary antibody. Concerning day 42 sera of group 1, the reactivity of 1:100 and 1:20 dilutions was also examined. To show the reactivity of anti-MPO heavy chain antibody to native MPO, a similar FCM was performed using the anti-MPO heavy chain monoclonal antibody (5 μg/ml; 4A4; Bio-Rad, Tokyo, Japan) as primary antibody and mouse IgG2b (5 μg/ml; BioLegend) as isotype control. b NET-forming neutrophils detected by FCM. Human peripheral blood neutrophils (1 × 106/ml) were treated with 5 ng/ml TNF-α for 15 min at 37 °C and then exposed to 10% rat sera. After incubation for 3 h at 37 °C, cells were next made to react with a plasma membrane-impermeable DNA-binding dye, SYTOX Green (Life Technologies, Carlsbad, CA, USA). After filtering out the debris with a mesh, the percolated cells were subjected for FCM. Histograms highlighted in green represent NET-forming neutrophils. The percentage of NET-forming neutrophils induced by group 2 sera was significantly higher than that induced by group 1 sera. c ANCA detected by immunoblotting. Lysates of human neutrophils boiled under reducing condition were electrophoresed (5 × 105 cells/lane) and then transferred to polyvinylidene difluoride membrane. After blocking the non-specific binding of antibodies, the membrane was incubated in diluted rat sera (day 42; group 1, 1:200 dilution; group 2, 1:1000 dilution) overnight at 4 °C. After rinsing with phosphate-buffered saline (PBS) with Tween 20 (PBS-T), the membrane was next incubated in the solution of horseradish peroxidase (HRP)-conjugated secondary antibody for 1 h at RT. After rinsing with PBS-T, the HRP activity on the membrane was detected by chemiluminescence using ImageQuant LAS 4000 (GE Healthcare, Little Chalfont, UK). Blue arrowhead, MPO heavy chain (59 kDa); red arrowheads, MPO light chain (14 kDa). d Degree of hematuria assessed at urine sampling immediately by a dipstick (Siemens Healthineers, Erlangen, Germany). e Degree of renal tissue damage. Erythrocyte casts (yellow arrowheads) were counted in the maximum longitudinal section of the kidney. f Degree of pulmonary hemorrhage. The foci of pulmonary hemorrhage were counted in the maximum longitudinal section of the lung. Mann-Whitney U test was applied for statistical analyses between two non-parametric groups

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