Alizarin Red Staining: A Technique to Visualize Mineralization by Cultured Osteoblasts
Published: April 30, 2023
Abstract
Source: Kampleitner, C., et al. Biological Compatibility Profile on Biomaterials for Bone Regeneration. J. Vis. Exp. (2018).
This video demonstrates a staining technique for detecting the extent of mineralization or calcium deposition in cultured primary osteoblasts using Alizarin Red dye.
Protocol
1. Osteoblast (OB) mineralization assessed by staining with Alizarin Red S (ARS)
- Add primary osteoblasts (OBs) to the β-tricalcium phosphate (β-TCP) disks used as a biocompatible scaffold in a culture plate.
- 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.
- Aspirate the 10% buffered formalin with a single-use pipet and wash twice with 0.5 mL of ultrapure water.
- 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.
- 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. - 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.
- Aspirate the PBS, transfer the stained β-TCP disks to a new well, and scan the plates with a flatbed scanner to record mineralization.
Divulgazioni
The authors have nothing to disclose.
Materials
| 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 |
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Citazione di questo articolo
Alizarin Red Staining: A Technique to Visualize Mineralization by Cultured Osteoblasts. J. Vis. Exp. (Pending Publication), e21048, doi: (2023).