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.
- 4% Formaldehyde
- 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)
- Rinse culture with PBS
- Fix cells with 4% Formaldehyde for 5 minutes at room temperature.
- Rinse with phosphatase buffered saline (PBS)
- If TRAcP staining is performed after cells were fixed, proceed directly to step 5
- Prepare Naphtol-AS-BI-phosphate solution
- 10mg/ml of Naphtol-AS-BI-phosphate in Dimethylformamide (Stable~2 weeks at 4°C)
- 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
- 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)
- Mix and filter (0.45mm) solution Aand Bin clean glass test tube (Staining Solution)
- Incubate cells for 50 to 60 minutes at 37°Cwith staining solution [Tip No. 5].
- Rinse twice with PBS
- Add 200ml of 70% ethanol to each well
- Wrap plate in cling film and store at 4°C
- Veronal buffer: mix 1.17g Sodium Acetate Anhydrous with 2.94g Veronal (sodium barbiturate) in 100ml dH2O and adjust pH to 10.1.
- 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.
- Acetate buffer (100mM Tartrate):add sodium tartrate to a concentration of 100 mM to the pH 5.2 acetate buffer.
- 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.
- Osteoclasts generated from primary cultures and cell lines may require different incubation period with staining solution.
- van’t Hof, R.J., 2003. Osteoclast formation in the mouse co-culture assay. Methods Mol. Med. 80, 145–152.
- 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.
- 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.
Department of Oncology and Metabolism, University of Sheffield, Sheffield, S10 2RX, UK. Email: firstname.lastname@example.org
Robert van’t Hof
Institute of Ageing and Chronic Disease, University of Liverpool, Liverpool, L7 8TX, UK.