3.4. Measurement of Cytokine Secretion by ELISA

ELISAs were carried out at the Cytokine Core Laboratory at the Center for Innovative Biomedical Resources (CIBR) Genomics Core Facility at the University of Maryland. To measure TNF-α cytokine production, we used enzyme-linked immunosorbent analysis (ELISA). Monocytic cells were cultured in 12-well plates at a density of 2 × 10⁵ cells/well in 1.2 mL of macrophage differentiation medium per well for 5–10 days. On the day of analysis, cells were washed in PBS, and macrophage medium was replaced with medium containing 3 nM recombinant human C5a (rC5a) (Sino Biological, Beijing, China #10604-HNAE). After 8 h, macrophage culture supernatants were collected and assayed for the indicated cytokines by ELISA as described below. After an 8 h rC5a (3 nM) incubation, supernatant was collected and submitted to the University of Maryland Cytokine Core Laboratory for analysis. Where indicated, the C5aR antagonist, PMX53 (50 nM), (Sigma) was added concurrently with rC5a during the 8 h incubation period. Where indicated, GD iPSC macrophages were incubated with recombinant human glucocerebrosidase (rGCase) (Cerezyme^®, Genzyme, Cambridge, MA, USA) at a concentration of 0.24 U/mL for 3 days prior to rC5a incubation. Cerezyme was obtained from patient infusion remnants. The cytokines were measured by two-antibody ELISA using biotin-strepavidin-peroxidase detection. Polystyrene plates Maxisorp (Nunc, Roskilde, Denmark) were coated with capture antibody in PBS overnight at 25 °C. The plates were washed 4 times with 50 mM Tris, 0.2% Tween-20, pH 7.0–7.5 and then blocked for 90 min at 25 °C with assay buffer (PBS containing 4% BSA (Sigma, St. Louis, MO, USA). Then, 50 µL of sample or standard prepared in assay buffer was added to each well and incubated at 37 °C for 2 h. The plates were washed 4 times and 100 µL of biotinylated detecting antibody in assay buffer was added and incubated for 1 h at 25 °C. After washing the plate 4 times, strepavidin-peroxidase polymer in casein buffer (Research Diagnostics Inc., Flanders, NJ, USA) was added and incubated at 25 °C for 30 min. The plate was washed 4 times and 100 µL of commercially prepared substrate (TMB; Dako, Carpinteria, CA, USA) was added and incubated at 25 °C for approximately 10–30 min. The reaction was stopped with 100 µL 2N HCl and the A450 (minus A650) was read on a microplate reader (Molecular Devices, San Jose, CA, USA). A curve was fitted to the cytokine standards using SoftMaxPro 7; Molecular Devices, San Jose, CA, USA) and cytokine concentration in each sample was calculated from the standard curve equation.