Resorption Pit Assay: An In Vitro Technique to Quantify Calcium Resorption Activity of Mature Osteoclasts using Calcium Phosphate-Coated Culture Plates

Protocolo

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

1. 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).
2. 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

1. To facilitate cell adhesion, incubate the calcium phosphate (CaP) coated plates with 50 μL of FBS for 1 h in a 37 °C incubator.
2. 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.
   1. 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.
   2. Determine the total cell number using a Neubauer chamber or similar.
3. 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.
4. Aspirate FBS solution from the CaP coated 96-well plate and pipette 200 μL of cell suspension per well (2 x 10⁵ cells/well).
5. 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.
6. At the end of incubation, wash cells with PBS twice, fix with 4% paraformaldehyde for 10 min, and wash with PBS again.
7. Use the fixed OCs directly for fluorescence staining or store at 4 °C.

3. Fluorescence staining of OCs and calcium phosphate coating

1. Incubate the fixed cells with permeabilization buffer (0.1% Triton in PBS) for 5 min.
2. 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.
3. Stain CaP coating with 100 μL of 10 μM calcein in PBS for 10 min. Wash three times with PBS and take images.

Materiais

Name	Company	Catalog Number	Comments
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
Pen-Strep	Lonza	DE17-602E	Penicillin-Streptomycin Mixture
Phalloidin-Alexa Fluor 546	Invitrogen	A22283	Alexa Fluor 546 Phalloidin
RANKL	PeproTech	310-01	Recombinant Human sRANK Ligand (E.coli derived)
Triton X-100	Sigma-Aldrich	T8787	Alkyl Phenyl Polyethylene Glycol
TrypLE Express	Gibco	12605010	Recombinant cell-dissociation enzymes