TRAcP Staining

Introduction

Tartrate-resistant acid phosphatase (TRAcP) is a reliable marker for identification of multinucleated osteoclasts and their precursor cells (Fig. 1). This HubLE Method describes the protocol for staining of TRAcP in fixed cell cultures.

Materials

• Dimethylformamide
• 4% Formaldehyde
• Naphtol-AS-BI-phosphate
• Veronal buffer [Tip No. 1]
• Acetate buffer [Tip No. 2 ]
• Acetate buffer (with Tartrate) [Tip No. 3]
• Pararosaniline [Tip No. 4]
• Sodium Nitrite (4%)
• Phosphate buffered saline (PBS)

Method [Update]

1. Rinse culture with PBS
2. Fix cells with 4% Formaldehyde for 5 minutes at room temperature.
3. Rinse with phosphatase buffered saline (PBS)
4. If TRAcP staining is performed after cells were fixed, proceed directly to step 5
5. Prepare Naphtol-AS-BI-phosphate solution

• 10mg/ml of Naphtol-AS-BI-phosphate in Dimethylformamide (Stable~2 weeks at 4°C)

6. Prepare staining Solution A: Mixthe following in a clean GLASStest tube;

• 150ml of Naphtol-AS-BI-phosphate
• 750ml of Veronal buffer
• 900ml of Acetate buffer

7. Prepare staining Solution B: Mix the following in a clean GLASS test tube;

• 120ml of Pararosaniline
• 120ml of Sodium Nitrite (4%) (Toxic cabinet) (Do not mix vigorously)

8. Mix and filter (0.45mm) solution Aand Bin clean glass test tube (Staining Solution)
9. Incubate cells for 50 to 60 minutes at 37°Cwith staining solution [Tip No. 5].
10. Rinse twice with PBS
11. Add 200ml of 70% ethanol to each well

• Wrap plate in cling film and store at 4°C

Tips [Update]

1. Veronal buffer: mix 1.17g Sodium Acetate Anhydrous with 2.94g Veronal (sodium barbiturate) in 100ml dH₂O and adjust pH to 10.1.
2. Acetate buffer: prepare solution A (Sol A, 0.82g Sodium Acetate Anhydrous in 100ml distilled water (dH₂O), and solution B (Sol B, 0.6ml glacial acetic acid made up to 100ml with dH₂ Adjust pH of solution A to 5.2 with solution B.
3. Acetate buffer (100mM Tartrate):add sodium tartrate to a concentration of 100 mM to the pH 5.2 acetate buffer.
4. Pararosaniline: add 1g to 20ml dH₂O and add 5ml concentrated HCl, Heat carefully in a water bath while stirring until fully dissolved do this in the fume cupboard. Filter after cooling to room temp.
5. Osteoclasts generated from primary cultures and cell lines may require different incubation period with staining solution.

References [Update]

1. van’t Hof, R.J., 2003. Osteoclast formation in the mouse co-culture assay. Methods Mol. Med. 80, 145–152.
2. Lv Y, Wang G, Xu W, Tao P, Lv X, Wang Y. Tartrate-resistant acid phosphatase 5b is a marker of osteoclast number and volume in RAW 264.7 cells treated with receptor-activated nuclear kappaB ligand. Exp Ther Med 2015; 9(1): 143-6.

Footnote:

• To cite this article: A.I. Idris. R. van’t Hof. TRAcP staining, HubLE Methods. 2.. HubLE Methods. 2. DOI: 10.13140/RG.2.2.25993.90720
• This protocol is for research use only and is not intended for use in human.

Lab Method BK15