PACAP38 Enhances BMDC Migration

Enhanced migration of BMDCs by PACAP38 treatment was assessed as described previously with modifications [34 (link)]. In brief, BMDCs treated with PACAP38 (1, 10, and 100 nM) [35 (link)] were placed in 100 μL in the upper chamber (transwell: 6.5-mm diameter, 5.0-mm pore size) according to manufacturer's instructions (Costar, #3421). We used SDF-1α (100 ng/mL, R&D System, positive control) in the lower chambers. For the PAC1 receptor blocking study, PACAP (6–38) (10 μM, Bachem, Switzerland) was also added to the lower wells [36 (link)]. The assays were conducted over a 4-hour incubation period at 37°C in a 5% CO₂ incubator. Because almost all cells stay at the lower side of the membrane after migration, quantification can be performed by simply counting these cells. Adhered cells at the lower side of the membranes were counted under the microscopy as previously described [34 (link)]. For assessing migrated CD34⁺ cells, cells were collected from the bottom of a transwell and assessed by flow cytometry as described previously [37 (link)].