Alizarin Red S Staining Protocol for Calcium Detection in Cells and Tissues

The protocol below details the use of alizarin red S (ARS) staining to identify and quantify calcium in cultured cells and tissue sections. ARS is a well-known and common histological stain for identifying calcium deposits in samples. This protocol is highly recommended for the analysis of osteogenic differentiation in osteoblasts or MSCs.

1.What Is Alizarin Red S and Why Is It Used for Calcium Detection?

Alizarin red S is a specific binding dye that binds to calcium and forms an orange-red complex that is visible under a light microscope. ARS is used in biological experiments to visualize and quantify calcium deposition in the formation of mineralized matrix in cells and tissues. For the purposes of this protocol, alizarin red S will be used to identify and measure the mineralization in mesenchymal stem cells (MSCs) and bone cells. See Figure 1 for a diagram of the assay.

2. How Alizarin Red S Binds Calcium: Principle of the Staining Assay

The underlying principle of ARS staining is based on the selective chelation of calcium ions by the sulfonyl group in ARS. Upon staining of the samples, the dye selectively binds to insoluble calcium salts (such as calcium phosphate) to form a localized red stain. The amount of ARS bound to the samples can be assessed visually or by extraction and quantification by absorbance at 405–550 nm.

Mechanism of Calcium Binding by Alizarin Red S Figure 1. Principle of Alizarin Red S Binds Calcium

3. Reagents and Equipment Needed for Alizarin Red S Staining

Chemicals and Reagents

Alizarin Red S (CAS 130-22-3) – an active dye that binds to calcium and serves as a visual marker and a quantifiable material in experiments
Distilled water (Milli-Q or equivalent)
0.1% Ammonium hydroxide — for adjusting pH
10% Acetic acid or 10% Cetylpyridinium chloride — for extraction
Phosphate-buffered saline (PBS)
4% Paraformaldehyde — for cell fixation

Equipment

pH meter or pH strips
Light microscope
Spectrophotometer (405–550 nm)
Cell culture plates or slides for tissue samples
Pipettes and tips
Filter (0.22 µm) — optional for live or fixed cells
Fume hood, gloves, lab coat

4. Preparation of Alizarin Red S Working Solution

Dissolve 2 grams of ARS powder in 100 mL of distilled water (2% w/v).
Adjust the pH to 4.1–4.3 with 0.1% ammonium hydroxide or dilute HCl.
Sterilize the solution by passing through a 0.22 µm filter if used for live or fixed cells.
Store at 4 °C and protect from light. Use within 1 month.

Workflow of Alizarin Red S Staining Procedure Figure 2. Alizarin Red S Staining Protocol

5. Preparing Cell and Tissue Samples for Alizarin Red S Staining

For Cultured Cells

Wash cells twice with PBS.
Fix with 4% paraformaldehyde for 15–20 minutes at room temperature.
Wash once with PBS to remove excess fixative.

For Tissue Sections

If paraffin-embedded, deparaffinize and rehydrate the slides.
Wash thoroughly with distilled water or PBS.

6. Alizarin Red S Staining Procedure

Add ARS working solution to cover the sample (e.g., 1 mL per well in a 24-well plate).
Incubate for 20–30 minutes at room temperature in the dark.
Gently wash samples with distilled water 3–5 times to remove free dye.
Visualize the red stain under a bright-field microscope.

7. Quantification of Alizarin Red S Staining

To quantify the amount of calcium in samples:

Add 1 mL of 10% acetic acid (or 10% cetylpyridinium chloride) to each stained well.
Incubate for 15–30 minutes at room temperature with shaking.
Transfer the extract to a microcentrifuge tube.
Centrifuge at 12,000 rpm for 10 minutes to pellet the debris.
Transfer the supernatant and measure absorbance at 405–550 nm.
Use a standard curve for quantitative analysis.

8. Troubleshooting Common Problems in Alizarin Red S Staining

Issue	Possible Cause	Suggested Solution
Weak staining	Low calcium content	Prolong culture time or enhance mineralization
High background	Inadequate washing	Increase washing steps or use fresh dye
Uneven staining	Uneven fixation or drying	Ensure uniform fixation and avoid drying
No signal in quantification	Incorrect pH or expired dye	Check pH of staining solution and replace dye

9. Safety Guidelines and Waste Disposal for ARS Staining

Use a fume hood when staining and extracting.
Handle paraformaldehyde, ARS, and ammonium hydroxide with PPE.
Collect dye waste and used fixatives in labeled biohazard waste containers.
Dispose according to institutional or local regulations.

Reference

Moriguchi, T., et al. "Elucidation of adsorption mechanism of bone-staining agent alizarin red S on hydroxyapatite by FT-IR microspectroscopy." Journal of colloid and interface science 260.1 (2003): 19-25.

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