Histopaque 1077 kit

The peripheral blood leucocytes of all family members (excluding the 16-week-old fetus) were separated from whole blood using a Histopaque-1077 kit (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany; cat. no. 10771) according to manufacturer's protocol. Genomic DNA samples were then extracted from the peripheral blood leucocytes of all family members (excluding the 16-week-old foetus) using the Qiagen QIAamp DNA Mini kit (Qiagen Inc., Valencia, CA, USA) according to manufacturer's protocol. A foetal genomic DNA sample was carefully extracted from the amniotic fluid of the mother using the QIAamp Circulating Nucleic Acid kit (Qiagen Inc.) according to the manufacturer's protocol. In addition, DNA samples collected from 200 patients in the same population that did not present with diagnostic features of Crouzon syndrome were used as controls. DNA concentration and purity was measured using a NanoDrop^™ ND-1000 spectrophotometer (Thermo Fisher Scientific, Inc., Waltham, MA, USA). Genomic DNA samples were preserved at -20˚C prior to use.

The peripheral blood from the vaccinated mice were used to acquire peripheral blood mononuclear cell (PBMC) single-cell suspensions using Histopaque-1077 kit (Sigma-Aldrich) based on density centrifugation. The cells were diluted in 2 ml culture medium containing the same concentration of stimulus as described above to the 12-well plate at 1×10⁶ cells per well. The plate was incubated at 37°C, 5% CO₂, 100% humidity for 36 h. Brefeldin A solution (eBioscience, Inc.) of 1 µl was added into the cell culture for 4 h to inhibit the cytokine transport. Cells were harvested and 100 µl of Reagent A was added (fixation medium, fix and perm cell permeablization reagent; Caltag Laboratories; Invitrogen Life Technologies) and incubated for 15 min at room temperature. Cells were washed, and the cell pellets resuspended, then adding 100 µl of Reagent B (permeabilization medium) and the 1 µl FITC conjugate anti-mouse IL-2, 1.25 µl PE conjugate IFN-γ and 20 µl PerCP-Cy5.5 conjugate TNF-α (eBioscience, Inc.) per million cells. Vortexed and incubated for 20 min in the dark at 4°C. Cells were wash and the cell pellets resuspend in 0.5 ml of cell staining buffer for analyzes by flow cytometry.