Journal: JOR Spine
Article Title: Successful fishing for nucleus pulposus progenitor cells of the intervertebral disc across species
doi: 10.1002/jsp2.1018
Figure Lengend Snippet: Nucleus pulposus cell viability and Tie2 expression negatively correlates with age. Flow cytometric analysis of human primary nucleus pulposus cells (NPCs) isolated using enzymatic digestion. (A) Percentage of Tie2 + NPCs related to age shows a steep decline after 25 years of age ( R 2 = 0.9489). (B) Gating by PI threshold reveals a strong decline in percentage of viable NPCs isolated by enzymatic intervertebral disc (IVD) digestion with age. (C) Tie2 + NPCs, isolated by flow‐activated cell sorting for Tie2. Primary refers to NPC that were sorted for negative (Tie2 − ) and positive (Tie2 + ) Tie2 cell populations and were included as controls. Tie2 + NPC were expanded in vitro for 7 days (expanded) in αMEM +10% (v/v) FBS in normoxic culture conditions either alone (−), or supplemented with either 10 ng/mL transforming growth factor beta (TGF‐β) or 100 ng/mL fibroblast growth factor 2 (FGF2) or subjected to hypoxic culture conditions. Fold increase in gene (mRNA) and protein (protein) expression levels were determined in Tie2 + NPCs by qPCR and flow cytometry, respectively in primary NPCs (Tie2 − and Tie2 + after sorting) and in expanded Tie2 + NPCs. mRNA and protein expression levels in primary Tie2 − NPC were set at 1. * P < .05 and # P < .005 as compared to normoxic expanded Tie2 + NPCs. (D) the capacity of sorted Tie2 + NPCs to form spherical colony forming units (CFU‐S), assessed using methylcellulose semi‐solid culture medium for 10 days, decreases with age
Article Snippet: Alexa Fluor 647 anti‐human CD202b Antibody (Biolegend, Cat. No. 334210, https://www.biolegend.com/en-gb/products/alexa-fluor-647-anti-human-cd202b-tie2-tek-antibody-5251 ) Allophycocyanin‐conjugated anti‐human Tie2clone No. Ms 83715 (R&D Systems, Cat. No. FAB3131A, https://www.rndsystems.com/products/human‐tie‐2‐apc‐conjugated‐antibody‐83715_fab3131a ) Allophycocyanin‐conjugated streptoavidin (BD Biosciences, Cat. No. 349024, http://www.bdbiosciences.com/us/reagents/research/antibodies‐buffers/second‐step‐reagents/avidinstreptavidin/apc‐streptavidin/p/349024 ) Anti‐mouse Tie2 biotinylated goat polyclonal antibody (R&D Systems, Cat. No. BAF007, https://www.rndsystems.com/products/goat-anti-mouse-igg-biotinylated-antibody_baf007 ) Anti‐Tie2/TEK Antibody, Millipore clone Ab33 (MERCK, Cat. No. 05‐584, http://www.merckmillipore.com/NL/en/product/Anti‐Tie2/TEK‐Antibody‐clone‐Ab33,MM_NF‐05‐584 ) BD FACS clean solution (Becton Dickinson, Cat. No. 340345, http://www.bdbiosciences.com/jp/services/ordersupport/information/20141107.jsp ) Caution : Causes skin irritation, severe eye irritation, it toxic to aquatic life.
Techniques: Expressing, Isolation, FACS, In Vitro, Flow Cytometry
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