Single-cell suspensions of osteoclasts were collected after passing through a 40 µm cell strainer (BD Bioscience), incubated with Fc receptor block (#101319; Biolegend) for 10 min, and then incubated with CoraLite 488–conjugated galectin-3 polyclonal antibody (rabbit anti-mouse antibody; CL488-14979; Proteintech; epitopes mapped throughout the full-length protein), eFluor 660–conjugated anti–galectin-3 monoclonal antibody (rabbit anti-mouse antibody; #50-5301-82; Thermo Fisher Scientific; clone M3/38, epitopes mapped within the N-terminal region), PE-conjugated anti–galectin-1 antibody (goat anti-mouse antibody; IC1245P; R&D), or the corresponding rabbit and goat isotype control (#31235; #31245; Invitrogen) in flow cytometry staining buffer (eBioscience) for 30 min at 4°C. For Mitotracker Green staining, cells were incubated with 100 nM Mitotracker Green (M7514; Invitrogen) in HBSS for 45 min. Then, cells were subjected to flow cytometry analysis on a FACS Canto II (BD Bioscience). Data analysis was carried out using FlowJo software.
Protocol detail
Osteoclast Galectin Expression Profiling
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40 µm cell strainer
Fc receptor block
flow cytometry staining buffer
Mitotracker Green
FACS Canto II
Anti antibody
Antibody
Buffer
Cells
Clone
Epitopes
Fc receptor
Flow cytometry
Galectin
Galectin 1
Galectin 3
Goat
Hbss
Isotype
Mouse
Osteoclasts
Protein
Rabbit
Corresponding Organization : Wuhan University
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Variable analysis
independent variables
- Cell strainer used to collect single-cell suspensions of osteoclasts
- Incubation with Fc receptor block
- Incubation with various antibodies (CoraLite 488-conjugated galectin-3 polyclonal antibody, eFluor 660-conjugated anti-galectin-3 monoclonal antibody, PE-conjugated anti-galectin-1 antibody)
- Incubation with Mitotracker Green
- Galectin-3 expression
- Galectin-1 expression
- Mitochondrial staining
- Flow cytometry staining buffer
- Corresponding rabbit and goat isotype controls
- Positive controls: not mentioned
- Negative controls: Rabbit and goat isotype controls
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