Osteoclast Characterization and Bone Resorption Assay
Osteoclasts cultured on plastic were stained using the Tartrate Resistant Acid Phosphatase (TRAP) Kit (Sigma-Aldrich), following the manufacturer's instructions. Osteoclasts differentiated on bone slices were stained using alendronate conjugated to Alexa Fluor 488,24 (link)TRITC-conjugated phalloidin (Sigma-Aldrich) and TO-PRO-3 (Thermo Fisher Scientific). To evaluate bone resorption, the same bone slices were used for toluidine blue staining as previously described.25 (link) TRAP and toluidine blue images were acquired on a Zeiss AxioImager M2m microscope, while immunofluorescence staining images were acquired on a Leica TCS SP5 Laser Scanning Confocal microscope. Quantification of CTX-I was performed on culture supernatants using CrossLaps for Culture (CTX-I) ELISA (Immunodiagnostic Systems), according to the manufacturer’s instructions.
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Capo V., Penna S., Merelli I., Barcella M., Scala S., Basso-Ricci L., Draghici E., Palagano E., Zonari E., Desantis G., Uva P., Cusano R., Sergi L.S., Crisafulli L., Moshous D., Stepensky P., Drabko K., Kaya Z., Unal E., Gezdiric A., Menna G., Serafini M., Aiuti A., Locatelli S.L., Carlo-Stella C., Schulz A.S., Ficara F., Sobacchi C., Gentner B, & Villa A. (2020). Expanded circulating hematopoietic stem/ progenitor cells as novel cell source for the treatment of TCIRG1 osteopetrosis. Haematologica, 106(1), 74-86.
Other organizations :
The San Raffaele Telethon Institute for Gene Therapy, Istituti di Ricovero e Cura a Carattere Scientifico, National Research Council, Humanitas University, Environment Park, Hôpital Necker-Enfants Malades, Hadassah Medical Center, Medical University of Lublin, Gazi University, Erciyes University, İstanbul Kanuni Sultan Süleyman Eğitim ve Araştırma Hastanesi, Sağlık Bilimleri Üniversitesi, Santobono Children's Hospital, University of Milano-Bicocca, Universität Ulm
Alexa Fluor 488-conjugated alendronate staining of osteoclasts differentiated on bone slices
TRITC-conjugated phalloidin staining of osteoclasts differentiated on bone slices
TO-PRO-3 staining of osteoclasts differentiated on bone slices
Toluidine blue staining of bone slices
Quantification of CTX-I in culture supernatants
control variables
Manufacturer's instructions for TRAP staining kit
Manufacturer's instructions for CrossLaps for Culture (CTX-I) ELISA
positive controls
Not specified
negative controls
Not specified
Annotations
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