Isolation of avian osteoclasts: Improved techniques to preferentially purify viable cells

Merry Jo Oursler, Patricia Collin-Osdoby, Fred Anderson, Ling Li, David Webber, Philip Osdoby

Research output: Contribution to journalArticle

40 Citations (Scopus)

Abstract

Among the many different methods that have been used to obtain and study isolated osteoclasts from a variety of species, the egg-laying hen maintained on a low-calcium diet has proven to be one of the richest sources of relatively large numbers of osteoclasts. However, recent reports and our own observations indicate that only a very small proportion of the osteoclasts harvested by such methods are viable.(1) The difficulty in obtaining large numbers of viable osteoclasts has restricted studies of osteoclast function and regulation, and so new isolation methods were sought. This report describes an osteoclast isolation procedure designed to substantially enrich for large numbers of viable authentic osteoclasts. Size and cell density differences between osteoclasts and contaminating mononuclear cells have been exploited in developing the methods for osteoclast enrichment. Sequential nonenzymatic and enzymatic procedures, followed by cell density separations, have yielded three populations of osteoclasts derived from chick hatchlings maintained on a low-calcium diet. A corresponding decrease in bone-associated osteoclasts during the sequential isolation scheme has been monitored using an osteoclast-directed monoclonal antibody, 121F. The first two populations contain 40% osteoclasts, which are predominantly (>99%) nonviable, but the third population contains 8-fold more viable osteoclasts, effectively increasing the proportion of viable osteoclasts more than 25-fold in comparison with the first two populations. The osteoclast-like nature of the isolated viable population 3 cells was established by demonstrating ruffled border formation, possession of the 121F monoclonal antibody-reactive osteoclast antigen, bone particle resorption activity, and resorption pit formation on cortical bone slices revealed by transmission and scanning electron microscopy.

Original languageEnglish (US)
Pages (from-to)375-385
Number of pages11
JournalJournal of Bone and Mineral Research
Volume6
Issue number4
StatePublished - Apr 1991
Externally publishedYes

Fingerprint

Osteoclasts
Population
Cell Count
Monoclonal Antibodies
Diet
Calcium
Scanning Transmission Electron Microscopy
Cell Separation
Bone Resorption

ASJC Scopus subject areas

  • Surgery

Cite this

Oursler, M. J., Collin-Osdoby, P., Anderson, F., Li, L., Webber, D., & Osdoby, P. (1991). Isolation of avian osteoclasts: Improved techniques to preferentially purify viable cells. Journal of Bone and Mineral Research, 6(4), 375-385.

Isolation of avian osteoclasts : Improved techniques to preferentially purify viable cells. / Oursler, Merry Jo; Collin-Osdoby, Patricia; Anderson, Fred; Li, Ling; Webber, David; Osdoby, Philip.

In: Journal of Bone and Mineral Research, Vol. 6, No. 4, 04.1991, p. 375-385.

Research output: Contribution to journalArticle

Oursler, MJ, Collin-Osdoby, P, Anderson, F, Li, L, Webber, D & Osdoby, P 1991, 'Isolation of avian osteoclasts: Improved techniques to preferentially purify viable cells', Journal of Bone and Mineral Research, vol. 6, no. 4, pp. 375-385.
Oursler, Merry Jo ; Collin-Osdoby, Patricia ; Anderson, Fred ; Li, Ling ; Webber, David ; Osdoby, Philip. / Isolation of avian osteoclasts : Improved techniques to preferentially purify viable cells. In: Journal of Bone and Mineral Research. 1991 ; Vol. 6, No. 4. pp. 375-385.
@article{301f76fe3d814d55a5606464e4b80ce1,
title = "Isolation of avian osteoclasts: Improved techniques to preferentially purify viable cells",
abstract = "Among the many different methods that have been used to obtain and study isolated osteoclasts from a variety of species, the egg-laying hen maintained on a low-calcium diet has proven to be one of the richest sources of relatively large numbers of osteoclasts. However, recent reports and our own observations indicate that only a very small proportion of the osteoclasts harvested by such methods are viable.(1) The difficulty in obtaining large numbers of viable osteoclasts has restricted studies of osteoclast function and regulation, and so new isolation methods were sought. This report describes an osteoclast isolation procedure designed to substantially enrich for large numbers of viable authentic osteoclasts. Size and cell density differences between osteoclasts and contaminating mononuclear cells have been exploited in developing the methods for osteoclast enrichment. Sequential nonenzymatic and enzymatic procedures, followed by cell density separations, have yielded three populations of osteoclasts derived from chick hatchlings maintained on a low-calcium diet. A corresponding decrease in bone-associated osteoclasts during the sequential isolation scheme has been monitored using an osteoclast-directed monoclonal antibody, 121F. The first two populations contain 40{\%} osteoclasts, which are predominantly (>99{\%}) nonviable, but the third population contains 8-fold more viable osteoclasts, effectively increasing the proportion of viable osteoclasts more than 25-fold in comparison with the first two populations. The osteoclast-like nature of the isolated viable population 3 cells was established by demonstrating ruffled border formation, possession of the 121F monoclonal antibody-reactive osteoclast antigen, bone particle resorption activity, and resorption pit formation on cortical bone slices revealed by transmission and scanning electron microscopy.",
author = "Oursler, {Merry Jo} and Patricia Collin-Osdoby and Fred Anderson and Ling Li and David Webber and Philip Osdoby",
year = "1991",
month = "4",
language = "English (US)",
volume = "6",
pages = "375--385",
journal = "Journal of Bone and Mineral Research",
issn = "0884-0431",
publisher = "Wiley-Blackwell",
number = "4",

}

TY - JOUR

T1 - Isolation of avian osteoclasts

T2 - Improved techniques to preferentially purify viable cells

AU - Oursler, Merry Jo

AU - Collin-Osdoby, Patricia

AU - Anderson, Fred

AU - Li, Ling

AU - Webber, David

AU - Osdoby, Philip

PY - 1991/4

Y1 - 1991/4

N2 - Among the many different methods that have been used to obtain and study isolated osteoclasts from a variety of species, the egg-laying hen maintained on a low-calcium diet has proven to be one of the richest sources of relatively large numbers of osteoclasts. However, recent reports and our own observations indicate that only a very small proportion of the osteoclasts harvested by such methods are viable.(1) The difficulty in obtaining large numbers of viable osteoclasts has restricted studies of osteoclast function and regulation, and so new isolation methods were sought. This report describes an osteoclast isolation procedure designed to substantially enrich for large numbers of viable authentic osteoclasts. Size and cell density differences between osteoclasts and contaminating mononuclear cells have been exploited in developing the methods for osteoclast enrichment. Sequential nonenzymatic and enzymatic procedures, followed by cell density separations, have yielded three populations of osteoclasts derived from chick hatchlings maintained on a low-calcium diet. A corresponding decrease in bone-associated osteoclasts during the sequential isolation scheme has been monitored using an osteoclast-directed monoclonal antibody, 121F. The first two populations contain 40% osteoclasts, which are predominantly (>99%) nonviable, but the third population contains 8-fold more viable osteoclasts, effectively increasing the proportion of viable osteoclasts more than 25-fold in comparison with the first two populations. The osteoclast-like nature of the isolated viable population 3 cells was established by demonstrating ruffled border formation, possession of the 121F monoclonal antibody-reactive osteoclast antigen, bone particle resorption activity, and resorption pit formation on cortical bone slices revealed by transmission and scanning electron microscopy.

AB - Among the many different methods that have been used to obtain and study isolated osteoclasts from a variety of species, the egg-laying hen maintained on a low-calcium diet has proven to be one of the richest sources of relatively large numbers of osteoclasts. However, recent reports and our own observations indicate that only a very small proportion of the osteoclasts harvested by such methods are viable.(1) The difficulty in obtaining large numbers of viable osteoclasts has restricted studies of osteoclast function and regulation, and so new isolation methods were sought. This report describes an osteoclast isolation procedure designed to substantially enrich for large numbers of viable authentic osteoclasts. Size and cell density differences between osteoclasts and contaminating mononuclear cells have been exploited in developing the methods for osteoclast enrichment. Sequential nonenzymatic and enzymatic procedures, followed by cell density separations, have yielded three populations of osteoclasts derived from chick hatchlings maintained on a low-calcium diet. A corresponding decrease in bone-associated osteoclasts during the sequential isolation scheme has been monitored using an osteoclast-directed monoclonal antibody, 121F. The first two populations contain 40% osteoclasts, which are predominantly (>99%) nonviable, but the third population contains 8-fold more viable osteoclasts, effectively increasing the proportion of viable osteoclasts more than 25-fold in comparison with the first two populations. The osteoclast-like nature of the isolated viable population 3 cells was established by demonstrating ruffled border formation, possession of the 121F monoclonal antibody-reactive osteoclast antigen, bone particle resorption activity, and resorption pit formation on cortical bone slices revealed by transmission and scanning electron microscopy.

UR - http://www.scopus.com/inward/record.url?scp=0025764147&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0025764147&partnerID=8YFLogxK

M3 - Article

C2 - 1858522

AN - SCOPUS:0025764147

VL - 6

SP - 375

EP - 385

JO - Journal of Bone and Mineral Research

JF - Journal of Bone and Mineral Research

SN - 0884-0431

IS - 4

ER -