Kaluza software

Th2 surface staining was performed as previously described (31 (link)). Briefly, 100 μl of whole blood was incubated with FITC-CD4 (SK3, BD, San Jose, CA, USA), PE-Cy5-CD183 (1C6/CXCR3, BD, San Jose, CA, USA), and PE-CD196 (11A9, BD, San Jose, CA, USA) at room temperature for 30 min in the dark. Isotype control was added to block the nonspecific binding. The cells were lysed using red blood cell lysate for 10 to 15 min and then washed with PBS twice. All samples were detected by BD flow cytometry (LSR II, BD, USA), and analyzed using Kaluza software (Beckman Coulter, Inc) or FlowJo_V10 (Tristar, USA).
ILC2 staining: 200 μl of whole blood was incubated with the following antibodies (all from BD unless specified otherwise), including Lineage-FITC, CD3 (UCHT1), CD19 (HIB19), CD123 (7G3), CD11b (M1/70), CD11c (B-ly6), CD8 (RPA-T8), FceRI (AER-37 (CRA-1), CD14 (M5E2), CD4 (RPA-T4), CD56 (B159), CD45-APC-Cy7 (2D1), CRTH2-PerCP-Cy5.5 (BM16), CD127-PE-Cy7 (HIL-7R-M21), at room temperature for 30 min in the dark. Isotype control was added to block the nonspecific binding. The cells were lysed using red blood cell lysate for 10 to 15 min and then washed with PBS twice. All samples were detected by BD flow cytometry (LSR II, BD, USA), and analyzed using Kaluza software (Beckman Coulter, Inc) or FlowJo_V10 (Tristar, USA).

UCB samples from each group were lysed twice using BD Pharm Lyse lysing buffer (BD Bioscience) at room temperature for 15 min and subsequently washed twice in phosphate-buffered saline with 2% fetal bovine serum to yield total nucleated cells (TNCs).
Staining for EPCs was performed with fluorescence-labeled antibodies for CD45 antigen (KO, Beckman Coulter), CD34 (PE-Cy7, BD Biosciences), CD133 (APC; clone CD133/1, Miltenyl Biotec), and KDR (known as VEGFR2; PE; clone 89106, R&D Systems). As a control, PE-conjugated isotype mouse IgG 1 was used. The absolute number of EPCs and the absolute number of white blood cells were calculated (individually for each sample) per 1 ml of UCB based on the percentage content of these cells as detected by flow cytometry (NAVIOS, Beckman Coulter Corp., Miami, FL, USA). The Kaluza software (Beckman Coulter) was used for analysis.
Staining for HSCs (CD34+) was performed with fluorescence-labeled antibodies for CD45 FITC/CD34 PE (Beckman Coulter) and 7-AAD Viability Dye (Beckman Coulter). As a control, CD45 FITC/IsoClonic PE-conjugated isotype was used. The number of HSCs were calculated (individually for each sample) per 1 ml UCB based on the content of these cells as detected by flow cytometry (NAVIOS, Beckman Coulter Corp., Miami, FL, USA). The Kaluza software (Beckman Coulter) was used for analysis.

Measurement of apoptotic cells was performed by detection of phosphatidylserine exposure using fluorescein isothiocyanate (FITC)-conjugated Annexin V (BD Biosciences, San Jose, CA, USA) and flow cytometry analysis (Gallios, Beckman Coulter, Miami, FL, USA), as described previously [66 (link)]. The percentage of FITC-Annexin V-positive cells was calculated as apoptotic cells using Kaluza software (Beckman Coulter, Miami, FL, USA). Intracellular fluorescence of Dox was detected by flow cytometry equipped with 488 nm excitation and 695 nm emission [67 (link)]. Fluorescent intensity was quantified with Kaluza software (Beckman Coulter, Miami, FL, USA) and represented as the mean fluorescent intensity (MFI).