Peripheral blood samples (2 mL each) were collected from the KD patients before IVIG and healthy children. PBMC were extracted using Ficoll-Paque PLUS (GE Healthcare Life Science) according to the manufacturer’s protocol. CECs were evaluated using a panel of antibodies: BV421-conjugated CD34 (BD Pharmingen, San Jose, CA, USA), PerCP/Cyanine5.5-conjugated CD45 (BioLegend, San Diego, CA, USA), phycoerythrin (PE)-conjugated CD133 (BioLegend), and Alexa Fluor® 647-conjugated CD144 (BioLegend). Alexa Fluor 488-conjugated EphB4 (R&D Systems, Minneapolis, MN, USA) was used to analyze the level of EphB4. CECs were identified as cells lacking CD45 expression, positive for CD34, positive for CD144, and negative for CD133 (CD45−/CD34+/CD144+/CD133−).
PBMC were blocked with TruStain FcX™ (BioLegend) for 10 min and then incubated with CD34, CD45, CD133, CD144, and Alexa Fluor 488-conjugated EphB4 (R&D Systems, MN, USA) for 15 min in the dark. Fluorescence intensity was examined by BD FACSLyric (BD) and analyzed by flow cytometry software FlowJoTM 10 (BD).
PBMC were blocked with TruStain FcX™ (BioLegend) for 10 min and then incubated with CD34, CD45, CD133, CD144, and Alexa Fluor 488-conjugated EphB4 (R&D Systems, MN, USA) for 15 min in the dark. Fluorescence intensity was examined by BD FACSLyric (BD) and analyzed by flow cytometry software FlowJoTM 10 (BD).