Igg1 pe

Phenotypic characterisation was performed on culture expanded MSCs at different passages and on cultured ECs at p4 using: CD31-FITC (#MCA1738F), CD105-PE (#MCA1557PE), CD90-PE (#MCA90PE) (all from Serotec, Kidlington, UK), CD73-PE (#550257), CD146-PE (#550315) (both from BD Pharmingen, Oxford, UK), and CD271-PE (#130-091-885, Miltenyi Biotec). The isotype controls were IgG1-FITC (#550616, BD Pharmingen) and IgG1-PE (#MCA928PE, Serotec). A total of 2×10
⁵ cells was stained with 5 μl FITC- or PE-conjugated antibodies, and dead cells were excluded using 2 μg/ml propidium iodide (PI, #P1304MP, Invitrogen). Cells were acquired using FACScan equipped with CellQuest software version 3.1 (BD Biosciences) and the proportions of the different fractions were calculated as a percentage of total live cells.

Cell sorting was performed using a MoFlo cell sorter equipped with SUMMIT software (Beckman Coulter, Buckinghamshire, UK). Following collagenase digestion of UC tissue, 2×10
⁶ cells were split into two tubes. One tube was stained with 5 μl of neat CD45-FITC (#F0861), CD235α-FITC (#F0870) (both from DAKO, Cambridge, UK), CD146-PE (BD Pharmingen) and CD31-APC (#130-092-652, Miltenyi Biotec), whereas the other was stained with 2.5 μl of neat isotype controls IgG1-FITC (#550617, BD Pharmingen), IgG1-PE (#MCA928PE, Serotec) and IgG1-APC (#130-098-846, Miltenyi Biotec). After incubation with relevant antibodies and washes, 2 μg/ml 7-aminoactinomycin D (7-AAD) (#A1310, Invitrogen) was added to exclude dead cells before sorting into four fractions: haemopoietic cell fraction (HC), CD45
⁺CD235α
⁺CD31
^-; EC fraction, CD45
^-CD235α
^-CD31
⁺; candidate MSC fraction, CD45
^-CD235α
^-CD31
^-CD146
⁺ and non-MSC fraction, CD45
^-CD235α
^-CD31
^-CD146
^-. The latter two subsets were processed for telomere length analysis.