Apoptosis of cells transfected with EGFP and control group cells was determined by staining with annexin V phycoerythrin
(PE) (Beyotime), and analyzed using FCM.
 Nonstandard abbreviations: VLBW, very low birth weight; NEC, necrotizing enterocolitis; LOS, late-onset sepsis; NICU, neonatal intensive care unit; PE, phycoerythrin
; PerCP, peridinin chlorophyll protein; BD, Becton Dickinson; FACS, fluorescence-activated cell sorter; CRP, C-reactive protein; AUROC, area under the ROC; WCC, white cell count.
Monoclonal antibodies coupled with phycoerythrin
(PE), peridinin-chlorophyll protein (PerCP), allophycocyanin (APC), and phycoerythrin-Cy-7 (PE-Cy7) were combined for simultaneous staining.
Fluorescein isothiocyanate (FITC)-conjugated anti-human CD16, phycoerythrin
(PE)-conjugated anti-human CD158a (EB6) and PE-conjugated anti-human CD158b (GL183) were purchased from Immunotech, Marseille, France.
The microarrays were stained with streptavidin phycoerythrin
(Molecular Probes, Eugene, OR, USA), washed again with 6x SSPE, stained with biotinylated antistreptavidin IgG followed by streptavidin phycoerythrin
, and washed a third time with 6x SSPE.
Abbreviations: FITC, fluorescein isothiocyanate; PE, phycoerythrin
; SSC, side scatter.
Fluorescein isothiocyanate (FITC)-conjugated mouse anti-human anti-ALDH1 monoclonal antibody, phycoerythrin
(PE)-conjugated mouse anti-human anti-CD133 monoclonal antibody, as well as the isotype control, were obtained from eBioscience (USA).
The antibodies labeled with phycoerythrin
(PE) were used: antihuman TNF RI (R&D systems, Minneapolis, MN, USA, cat number FAB225P, clone 16803.1, mouse IgG1) and antihuman TNF RII (R&D systems, cat number FAB226P, clone 22235, mouse IgG2A).
Threecolor staining--fluorescein-isothiocyanate, phycoerythrin
, and allophycocyanin-conjugated primary antibodies against ABCG2, CD31, CD34, CD44, CD47, CD49a, CD49d, CD51, CD73, CD90, CD105, and Nestin--were applied for 30 minutes at 4[degrees]C.
All FITC-, phycoerythrin
(PE)-, and PE-Cy5 conjugated antibodies except for BDCA-2 were purchased from Biolegend (London, UK).
Mice were then euthanized on the indicated days, and 5 [micro]l blood was stained with streptavidin conjugated with phycoerythrin
(BD Pharmingen) and TER119 mAb conjugated with fluorescein isothiocyanate.
Luminex 100 flow analyzer detected the fluorescent intensities of Phycoerythrin
(PE) on each microbeads.