Resorption Pit Assay: An In Vitro Technique to Quantify Calcium Resorption Activity of Mature Osteoclasts using Calcium Phosphate-Coated Culture Plates
In this video, we describe an in vitro osteoclastic resorption assay on calcium phosphate-coated cell culture plates cultured with human peripheral blood mononuclear cell-derived osteoclasts. The osteoclasts form a characteristic actin ring on the calcium phosphate-coated matrix of the cell culture plate and degrade the matrix within the actin ring region to release calcium and phosphate ions, resulting in the formation of distinct resorption pits within the degraded matrix.
Protocol
All procedures involving human participants have been performed in compliance with the institutional, national, and international guidelines for human welfare and have been reviewed by the local institutional review board.
1. Expansion of OC progenitors
Resuspend PBMCs in complete α-MEM (10% FBS, 1% Pen/Strep, 1% amphotericin B) containing 20 ng/mL M-CSF. Seed PBMCs at a density of 2.5 x 105 cells/cm2. Usually, cells isolated from 15-20 mL fresh blood can be seeded in one 75 cm2 flask (1.5-2 x 107 cells).
Feed the cells with fresh complete α-MEM containing 20 ng/mL M-CSF every third day until the attached cells reach a desired confluency. Typical yields are 1.5-2 x106 cells/flask when cells are 95% confluent. Usually, the expansion period lasts 6 days. NOTE: The osteoclastogenic potential of the precursors can decrease with prolonged cultivation time.
2. Induction of osteoclastogenesis in calcium phosphate coated plates
To facilitate cell adhesion, incubate the calcium phosphate (CaP) coated plates with 50 μL of FBS for 1 h in a 37 °C incubator.
To detach the OC precursors, wash the flask with PBS twice to remove dead or non-adherent cells. Add 4 mL of trypsin (Table of Materials) per 75 cm2 flask, for 30 min.
Add 4 mL of complete α-MEM to stop the digestion, carefully detach the cells using a cell scraper and transfer the cells into a 50 mL tube.
Determine the total cell number using a Neubauer chamber or similar.
Pellet the cells by centrifugation for 7 min at 350 x g. Resuspend the pellet in sufficient complete α-MEM containing 20 ng/mL M-CSF and 20 ng/mL RANKL to get a concentration of 1 x 106 cells/mL.
Aspirate FBS solution from the CaP coated 96-well plate and pipette 200 μL of cell suspension per well (2 x 105 cells/well).
Incubate the OC precursors for the desired time period or with the desired reagents relevant to the experimental design. Usually, high numbers of large and multinucleated OCs can be observed after 6 days and when resorption pits are forming. Feed cells with fresh complete α-MEM medium containing 20 ng/mL M-CSF and 20 ng/mL RANKL every third day.
At the end of incubation, wash cells with PBS twice, fix with 4% paraformaldehyde for 10 min, and wash with PBS again.
Use the fixed OCs directly for fluorescence staining or store at 4 °C.
3. Fluorescence staining of OCs and calcium phosphate coating
Incubate the fixed cells with permeabilization buffer (0.1% Triton in PBS) for 5 min.
Stain actin filaments with 100 μL of AlexaFluor 546 labeled phalloidin solution in PBS for 30 min and aspirate the staining solution. Add 100 μL of Hoechst 33342 staining solution (10 μg/mL in PBS) for 10 min to stain nuclei. DAPI can also be used.
Stain CaP coating with 100 μL of 10 μM calcein in PBS for 10 min. Wash three times with PBS and take images.
Declarações
The authors have nothing to disclose.
Materials
α-MEM
Gibco
32561-029
MEM alpha, GlutaMAX, no nucleosides
amphotericin B
Biochrom
03-028-1B
Amphotericin B Solution
Calcein
Sigma-Aldrich
C0875
Calcein
FBS
Sigma-Aldrich
F7524
fetal bovine serum
Fixation buffer
Biolegend
420801
Paraformaldehyde
Hoechst 33342
Promokine
PK-CA707-40046
Hoechst 33342
M-CSF
PeproTech
300-25
Recombinant Human M-CSF
PBS
Lonza
17-512F
Dulbecco's Phosphate Buffered Saline (1X), DBPS without Calcium and Magnesium
Resorption Pit Assay: An In Vitro Technique to Quantify Calcium Resorption Activity of Mature Osteoclasts using Calcium Phosphate-Coated Culture Plates. J. Vis. Exp. (Pending Publication), e21160, doi: (2023).