human osteosarcoma derived osteoblast like cell line  (ATCC)

Journal: Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research

Article Title: Intercellular adhesion molecule 1 discriminates functionally different populations of human osteoblasts: characteristic involvement of cell cycle regulators.

doi: 10.1359/jbmr.2000.15.10.1912

Figure Lengend Snippet: FIG. 1. ICAM-1 divided osteoblastic cells into two sub- populations. (A) Staining and flow cytometric analysis of human osteoblastic cells and human osteosarcoma-derived osteoblast-like cell line MG63 were carried out with control mAb thy-1.2, ICAM-1 (CD54) mAb 84H10, and VCAM-1 (CD106) mAb 2G7 using FACScan. The ordinate repre- sents the number of cells stained with mAbs in each loga- rithmic scale of fluorescence amplifier. The histogram of human osteoblastic cells is a representative example of five osteoarthritis patients. Dotted line represents the gate set to discriminate negative from positive stained cells as deter- mined by control thy-1.2 mAb. (B) Adhesion of monocytes to osteoblasts. Adhesion of PMA-activated peripheral nor- mal CD141 monocytes, which were labeled with 51Cr to unsorted, ICAM-11 or ICAM-12 osteoblastic cells was assessed in the presence or absence of anti–LFA-1 (CD18) mAb TS2/18, control anti-MHC class I mAb W6/32, or anti-ODF mAb no. 412 (at 10 mg/ml each) after 30 minutes incubation at 37°C. The g-emission of lysates of adherent cells only was determined. Data are expressed as mean percentage 6 SEM of binding of added monocytes and are representative results of five similar experiments.

Article Snippet: When cell monolayers reached confluence after a 6- to 8-week culture period, the explants were removed and the cells were replated and incubated, which resulted in new cellular outgrowth and eventually a confluent monolayer of cells, which possessed characteristics of osteoblast-like phenotype including osteocalcin, bone sialoprotein, type I collagen, and ALP, as previously described(5) The human osteosarcoma–derived osteoblast-like cell line MG63 (American Type Culture Collection [ATCC], Rockville, MD, U.S.A.) was grown in DMEM with 10% FCS.

Techniques: Staining, Derivative Assay, Control, Labeling, Incubation, Binding Assay

Journal: Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research

Article Title: Intercellular adhesion molecule 1 discriminates functionally different populations of human osteoblasts: characteristic involvement of cell cycle regulators.

doi: 10.1359/jbmr.2000.15.10.1912

Figure Lengend Snippet: FIG. 3. Osteoclast-like cells were induced from peripheral monocytes in the coculture with ICAM-11 osteoblastic cells or MG63. Purified CD141 peripheral monocytes were added to (A) ICAM-11 osteoblasts or (B and C) MG63 and they were cocultured for 2 weeks in DMEM with 20% FCS in the presence of 1027 M 1a,25(OH)2VD3. (A and B) Cathepsin K and (C) integrin avb3 were stained with anti-cathepsin K Ab and anti-avb3 mAb 23C6, and subsequent second FITC-conjugated anti-goat Ig or anti-mouse Ig, respectively, and were observed by confocal microscopy (31000).

Article Snippet: When cell monolayers reached confluence after a 6- to 8-week culture period, the explants were removed and the cells were replated and incubated, which resulted in new cellular outgrowth and eventually a confluent monolayer of cells, which possessed characteristics of osteoblast-like phenotype including osteocalcin, bone sialoprotein, type I collagen, and ALP, as previously described(5) The human osteosarcoma–derived osteoblast-like cell line MG63 (American Type Culture Collection [ATCC], Rockville, MD, U.S.A.) was grown in DMEM with 10% FCS.

Techniques: Staining, Confocal Microscopy

Journal: Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research

Article Title: Intercellular adhesion molecule 1 discriminates functionally different populations of human osteoblasts: characteristic involvement of cell cycle regulators.

doi: 10.1359/jbmr.2000.15.10.1912

Figure Lengend Snippet: FIG. 5. Expression levels of p21, p53, Rb, and c-Myc in ICAM-11 and ICAM-12 osteoblasts detected by specific Abs. ICAM-1-MG63 were harvested and solubilized in lysis buffer by sonication. Postnuclear supernatants were prepared by centrifugation and protein was quantitated. For Western blot analysis, samples containing equal amounts of proteins were subjected to SDS-PAGE. Separated proteins were transferred onto PVDF membranes. After blocking, membranes were incubated with anti-p21, anti-p53, anti-Rb, anti-c-Myc, anti-cdk2, and control antiactin Abs. After washing, the membranes were incubated with HRP- conjugated Abs and proteins were detected using a chemi- luminescence reagent.

Article Snippet: When cell monolayers reached confluence after a 6- to 8-week culture period, the explants were removed and the cells were replated and incubated, which resulted in new cellular outgrowth and eventually a confluent monolayer of cells, which possessed characteristics of osteoblast-like phenotype including osteocalcin, bone sialoprotein, type I collagen, and ALP, as previously described(5) The human osteosarcoma–derived osteoblast-like cell line MG63 (American Type Culture Collection [ATCC], Rockville, MD, U.S.A.) was grown in DMEM with 10% FCS.

Techniques: Expressing, Lysis, Sonication, Centrifugation, Western Blot, SDS Page, Blocking Assay, Incubation, Control

Journal: Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research

Article Title: Intercellular adhesion molecule 1 discriminates functionally different populations of human osteoblasts: characteristic involvement of cell cycle regulators.

doi: 10.1359/jbmr.2000.15.10.1912

Figure Lengend Snippet: FIG. 6. The cdk assay of osteoblasts. (A) ICAM-11 and ICAM-12 MG63 were solubilized in lysis buffer by sonication. Postnuclear supernatants were precleared with protein G-Sepharose and immunoprecipitated with anti-cdk6 Ab and protein G-Sepharose for 3 h at 4°C. Immunoprecipitates were washed four times with lysis buffer and then washed once with kinase reaction buffer. Immunoprecipitates were suspended in 40 ml of kinase buffer containing 0.5 mg of recombinant p110Rb protein and were incubated for 30 minutes at 37°C. The reactions were terminated by the addition of Laemmli sample buffer. Subsequently, samples were separated by SDS-PAGE under reducing conditions. The p110Rb band was visualized by staining of gels with Coomassie Brilliant Blue (R-250). (B) Gels were subjected to autoradiography and band intensities were quantitated using a Fujix imaging analyzer. Relative cdk6 activity of ICAM-11 MG63 was calculated and compared with that of ICAM-12 MG63.

Article Snippet: When cell monolayers reached confluence after a 6- to 8-week culture period, the explants were removed and the cells were replated and incubated, which resulted in new cellular outgrowth and eventually a confluent monolayer of cells, which possessed characteristics of osteoblast-like phenotype including osteocalcin, bone sialoprotein, type I collagen, and ALP, as previously described(5) The human osteosarcoma–derived osteoblast-like cell line MG63 (American Type Culture Collection [ATCC], Rockville, MD, U.S.A.) was grown in DMEM with 10% FCS.

Techniques: Lysis, Sonication, Immunoprecipitation, Recombinant, Incubation, SDS Page, Staining, Autoradiography, Imaging, Activity Assay