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)

  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.

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).

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