Catalog no 14190136

First, approximately 1 × 10⁵ cells were collected from the culture, centrifuged for 5 min at 300 g to remove the supernatant, and washed twice with 2 ml 1 × Dulbecco’s phosphate-buffered saline (DPBS; catalog No. 14190136, Gibco) solution containing 2% fetal bovine serum (Catalog No. 16000044, Gibco) and 2 mM EDTA. After centrifugation, the cells were resuspended in 50 μl of 1 × DPBS buffer, and the antibodies were added, followed by incubation at 4 °C for 10 min in the dark. The cells were washed twice by adding 2 ml of 1 × DPBS buffer after incubation and then resuspended in 200 μl of 1 × DPBS buffer to prepare a cell suspension for the assay. The BD FACSAria II instrument was used for flow cytometry analysis, and the data analysis was performed with Flowjo software (v.7.6, Three Star). The antibodies used in this study included anti-human CD34-FITC (Catalog No. 130-098-142, Miltenyi Biotec, Bergisch Gladbach, Germany), anti-human CD45-PE (Catalog No. 170-081-061, Miltenyi Biotec), anti-human CD71-PE (Catalog No. 130-099-219, Miltenyi Biotec), and anti-human CD235a-APC (Catalog No. 130-118-493, Miltenyi Biotec).

TV, LV, and RP cells from six consecutive vertebrae (three TV and three LV segments) close to the TLV segmentation joint of one LW pig embryo at 27 dpf were collected by micromanipulation and enzyme digestion. TV, LV, and RP cells were uniformly dissected into millimeter-sized pieces in Dulbecco’s phosphate-buffered saline (DPBS; Catalog No. 14190136, Gibco, Carlsbad, CA) supplemented with 10% fetal calf serum, transferred to tubes containing 1 ml of collagenase II (5 mg/ml; Catalog No. C5138-1G, Sigma, St. Louis, MI) and 1 ml of dispase (2.5 mg/ml; Catalog No. 42613–33-2, Sigma), and then incubated at 37 °C for 3–5 min. Digested tissue pieces were then filtered through a 40-mm nylon cell strainer (Catalog No. 352340, BD Falcon, Franklin, NJ) and centrifuged at 500 g for 10 min at 37 ℃ to collect cell pellets. The cell pellets were next washed using 1 × DPBS three times to remove fragments and then resuspended in Dulbecco’s Modified Eagle’s Medium (Catalog No. 11995040, Gibco).