The lymphocytes gated in the side-scatter/ CD45 dot-plot were analyzed for CD3+/CD4+, CD3+/CD8+, CD3-/CD19+ and CD3-/CD16/56+ populations using fluorescein isothiocyanate (FITC)-labeled CD4 (clone SK3), phycoerythrin
(PE)-labeled CD8 (clone SKI), APC-labeled CD3 (clone SK7) or APC-labeled CD19 (clone SJ25C1), FITC-labeled CD3, and PE-labeled CD 16 and PE-labeled CD56 panels on a FACSCalibur (all from BD Biosciences) equipped with CellQuest.
The cells were incubated with a series of monoclonal antibodies (Mabs) directly labeled with fluorescein isothiocyanate (FITC) or phycoerythrin
The 1st fraction was incubated with antibodies to CD45-Alexa 488 (AbD Serotec) and phycoerythrin
(PE)-conjugated CD34 (Santa Cruz Biotechnology), the 2nd fraction was incubated with CD45-Alexa 488 and isotype-PE for CD34 antibody, and the 3rd fraction was incubated with CD45-Alexa 488 and CD133-PE (Miltenyi Biotec) for 10-20 min at 4 [degrees]C.
Thus, the ASPE target typically contained several biotin moieties, which were subsequently visualized as a result of a binding reaction with phycoerythrin-labeled streptavidin (phycoerythrin
is a pigment-carrying, highly fluorescent protein from red algae).
Alternatively, cells were stained with fluorescein isothiocyanate-conjugated CD3 (BD Biosciences), CD19 tandemly conjugated with phycoerythrin
and Texas Red (Beckman Coulter), CD5 tandemly conjugated with phycoerythrin
and Cy7 (Beckman Coulter), and allophycocyanin-conjugated CD56 (Beckman Coulter) and analyzed on a FC500 instrument with CXP software (Beckman Coulter).
Strong AF was mainly observed in 488- and 561nm channels, thus interfering with the analysis of fluorescence emitted by the commonly used fluorophores, such as fluorescein isothiocyanate, tetramethylrhodamine, phycoerythrin
, Alexa Fluor 488, and Alexa Fluor 594.
The antibodies used for activating fluorescence and cell sorting were CD138/SYNDECAN-1 (cat: 347216) allophycocyanin, CD38 (cat: 340909) fluorescein isothiocyanate, CD19 (cat: 345777) phycoerythrin
, CD56 (cat: 557747), and phycoerythrin
cyanin (Becton Dickinson).
The colour arises from the photosynthetic pigment Phycoerythrin
. (7) Unlike other algae within the Rhodophytes, they form a solid carbonate structure.
In the first tube, cells were labelled using phycoerythrin
(PE) conjugated monoclonal antibody (MoAb)for surface markers CD3 (BD Biosciences Immunocytometry Systems, San Jose, CA) and fluorescein isothiocyanate (FITC) conjugated antibody for CD25 (BD Biosciences Immunocytometry Systems, San Jose, CA).
The resolution limit of the Apogee A60-Micro was 74 phycoerythrin
(PE) molecules ofequivalent soluble fluorochrome (MESF), 304 FITC MESF, and 16 APC MESF.
Abbreviations IBD: Inflammatory bowel disease DSS: Dextran sulfate sodium TJs: Tight junction proteins JAM-1: Junctional adhesion molecule 1 Foxp3: Forkhead box P3 IL-17: Interleukin-17 IL-10: Interleukin-10 DAI: Disease activity index H&E: Hematoxylin-eosin TNF[alpha]: Tumor necrosis factor PMSF: Phenylmethylsulfonyl fluoride TEM: Transmission electron microscopy FITC: Fluorescein isothiocyanate PE: Phycoerythrin
SEM: Standard error of the mean.
MAbs labeled with different fluorophores, i.e., FITC (fluorescein isothiocyanate), PE (phycoerythrin
), APC (allophycocyanin), PerCP-eFluor 710 (peridinin-chlorophyll-proteineFluor 710), and PE-Cy7 (phycoerythrin-cyanin7), were used.