Eia kit

Basal and stimulated extracellular CGRP concentrations were evaluated in freshly isolated DRG neurons using an EIA kit (Bachem) following the manufacturer’s instructions. The isolated cells were maintained in DMEM/F12 with 10% FBS for 2–3 h in a 24-well plate before EIA experiments. For each well, cells were washed three times with normal external solution and then incubated with normal or Ca²⁺-free external solution for 5 min at room temperature, followed by another 5-min incubation with the same solution containing 100 µM AITC. The incubating solutions were collected for subsequent analysis of basal and stimulation-coupled CGRP levels. A similar protocol was used for 100 mM K⁺-induced CGRP release from DRG neurons in normal external solution. All samples were centrifuged at 13,000 rpm for 5 min and the supernatants processed for CGRP measurement. Samples were analyzed at 450 nm with a microplate reader (Synergy 4; BioTek). CGRP concentrations (in picograms per milliliter) were extrapolated from a best-fit line calculated from serial dilutions of a CGRP standard. All data points were measured in triplicate.

Serum and retinal level of Ang-(1–7) was measured using a commercial EIA kit (Bachem, San Carlos, CA), following the manufacturer's instructions. All measurements were performed in duplicate and the data represent the mean of two separate assay results.