TY - JOUR
T1 - High throughput, quantitative analysis of human osteoclast differentiation and activity
AU - Diepenhorst, Natalie
AU - Nowell, Cameron
AU - Rueda, Patricia
AU - Henriksen, Kim
AU - Pierce, Tracie
AU - Cook, Anna
AU - Pastoureau, Philippe
AU - Sabatini, Massimo
AU - Charman, William
AU - Christopoulos, Arthur
AU - Summers, Roger
AU - Sexton, Patrick
AU - Langmead, Christopher
PY - 2017/2/15
Y1 - 2017/2/15
N2 - Osteoclasts are multinuclear cells that degrade bone under both physiological and pathophysiological conditions. Osteoclasts are therefore a major target of osteoporosis therapeutics aimed at preserving bone. Consequently, analytical methods for osteoclast activity are useful for the development of novel biomarkers and/or pharmacological agents for the treatment of osteoporosis. The nucleation state of an osteoclast is indicative of its maturation and activity. To date, activity is routinely measured at the population level with only approximate consideration of the nucleation state (an ‘osteoclast population’ is typically defined as cells with ≥3 nuclei). Using a fluorescent substrate for tartrate-resistant acid phosphatase (TRAP), a routinely used marker of osteoclast activity, we developed a multi-labelled imaging method for quantitative measurement of osteoclast TRAP activity at the single cell level. Automated image analysis enables interrogation of large osteoclast populations in a high throughput manner using open source software. Using this methodology, we investigated the effects of receptor activator of nuclear factor kappa-B ligand (RANK-L) on osteoclast maturation and activity and demonstrated that TRAP activity directly correlates with osteoclast maturity (i.e. nuclei number). This method can be applied to high throughput screening of osteoclast-targeting compounds to determine changes in maturation and activity.
AB - Osteoclasts are multinuclear cells that degrade bone under both physiological and pathophysiological conditions. Osteoclasts are therefore a major target of osteoporosis therapeutics aimed at preserving bone. Consequently, analytical methods for osteoclast activity are useful for the development of novel biomarkers and/or pharmacological agents for the treatment of osteoporosis. The nucleation state of an osteoclast is indicative of its maturation and activity. To date, activity is routinely measured at the population level with only approximate consideration of the nucleation state (an ‘osteoclast population’ is typically defined as cells with ≥3 nuclei). Using a fluorescent substrate for tartrate-resistant acid phosphatase (TRAP), a routinely used marker of osteoclast activity, we developed a multi-labelled imaging method for quantitative measurement of osteoclast TRAP activity at the single cell level. Automated image analysis enables interrogation of large osteoclast populations in a high throughput manner using open source software. Using this methodology, we investigated the effects of receptor activator of nuclear factor kappa-B ligand (RANK-L) on osteoclast maturation and activity and demonstrated that TRAP activity directly correlates with osteoclast maturity (i.e. nuclei number). This method can be applied to high throughput screening of osteoclast-targeting compounds to determine changes in maturation and activity.
KW - Bone resorption
KW - High throughput
KW - ImageJ
KW - Osteoclast
KW - Quantitative image analysis
KW - Tartrate-resistant acid phosphatase (TRAP)
UR - http://www.scopus.com/inward/record.url?scp=85007201355&partnerID=8YFLogxK
U2 - 10.1016/j.ab.2016.12.010
DO - 10.1016/j.ab.2016.12.010
M3 - Article
AN - SCOPUS:85007201355
SN - 0003-2697
VL - 519
SP - 51
EP - 56
JO - Analytical Biochemistry
JF - Analytical Biochemistry
ER -