Alizarin Red Staining: A Technique to Visualize Mineralization by Cultured Osteoblasts

Protocolo

1. Osteoblast (OB) mineralization assessed by staining with Alizarin Red S (ARS)

1. Add primary osteoblasts (OBs) to the β-tricalcium phosphate (β-TCP) disks used as a biocompatible scaffold in a culture plate.
2. Aspirate the culture medium from the wells containing primary OBs 14 days after the addition of the mineralization medium and rinse twice with 0.5 mL of 1x PBS at RT. Aspirate the PBS and fix the cells by adding 0.5 mL of the 10% buffered formalin solution at RT for 10 min.
3. Aspirate the 10% buffered formalin with a single-use pipet and wash twice with 0.5 mL of ultrapure water.
4. To the fixed OBs, add 0.25 mL of 40 mM ARS staining solution (pH 4.2) dissolved in ultrapure water and incubate the plate at RT for 10 min on a shaker at 100 shakes/min.
5. Aspirate the staining solution with a single-use pipet and rinse with 1 mL of ultrapure water. Repeat this step 5–10 times to remove unspecific staining.
   NOTE: The rinsing solution should be without color.
6. Aspirate the ultrapure water and add 1 mL of cold PBS. Incubate the plate at RT for 10 min on a shaker at 100 shakes/min.
7. Aspirate the PBS, transfer the stained β-TCP disks to a new well, and scan the plates with a flatbed scanner to record mineralization.

Materiales

Name	Company	Catalog Number	Comments
β-tricalcium phosphate disks (β-TCP, 14 mm)			Disks were sintered in a muffle furnace at a temperature of 1150 °C. A detailed description of the production can be found in Zimmer et al (doi: 10.1002/jbm.a.34639). Samples were UV-irradiated (15 min for each side) before using in cell cultures.
Orthovita Vitoss foam (β-TCP/C foam)	Orthovita	2102-1405
Alizarin Red S	Sigma-Aldrich	A5533
Formalin solution neutral buffered 10%	Sigma-Aldrich	HT501128
Osteogenic mineralization medium (MM)			α-MEM + 10% heat-inactivated FBS + 1% penicillin-streptomycin + 50 µg/mL ascorbic acid + 5 mM β-glycerophosphate
Phosphate-buffered saline 1x (PBS) pH 7.4	Gibco, Thermo Fisher Scientific	10010023
Ultrapure water: Cell culture water pyrogen free	VWR	L0970-500
Wash buffer			Add  0.05% Tween20 to 1x PBS.
24-well suspension culture plate	Greiner bio-one	662102
Flatbed scanner: Epson Perfection 1200 Photo	Epson
Single-use pipet: serum pipet	Greiner bio-one	612301
Distilled water	Carl Roth	3478.4
Single-use pipet: serum pipet	Greiner bio-one	612301