Materials and Reagents

Osteosarcoma cells (Basu-Roy et al., 2012)
DMEM - high Glucose from Invitrogen (Life Technologies, Invitrogen™, catalog number: 11330-032 )
Fetal bovine serum (FBS)
Penicillin-streptomycin
Beta-glycerol phosphate (Sigma-Aldrich, catalog number: G9422 )
Ascorbic acid (Sigma-Aldrich, catalog number: A4034 )
Earle's balanced salt solution (EBSS) from Invitrogen (Life Technologies, Invitrogen™, catalog number: 14155-063 )
HCl
Ethanol
DMSO (Sigma-Aldrich, catalog number: D8418 )
Phosphate buffered saline (PBS)
Paraformaldehyde
Isopropanol
Acetone
Double-distilled H₂O (ddw)
Citrate concentrated solution (20 ml) (Sigma-Aldrich, catalog number: 854C )
Naphthol AS-MX phosphate alkaline solution, 0.25% (20 ml) (Sigma-Aldrich, catalog number: 855 )
COMPLETE capsule of Fast Blue RR salt (Sigma-Aldrich, catalog number: FBS25-10 CAP )
Oil-Red-O (Sigma-Aldrich, catalog number: O0625 )
Mayer's HemTox stain (Sigma-Aldrich, catalog number: MHS1 )
Alizarin Red S certified by the biological stain commission (Sigma-Aldrich, catalog number: A5533 )
Beta-glycerol phosphate stock solution (see Recipes)
1,000x ascorbic acid solution (see Recipes)
Dexamethasone (Sigma-Aldrich, catalog number: D4902 ) (see Recipes)
3-Isobutyl-1-methylxanthine (IBMX) (Sigma-Aldrich, catalog number: I5879 ) (see Recipes)
Indomethacin (Sigma-Aldrich, catalog number: I8280 ) (see Recipes)
Insulin (recombinant human) (Sigma-Aldrich, catalog number: I2643 ) (see Recipes)
Fixation solution (see Recipes)
Staining solution for osteogenesis (see Recipes)
Oil-Red-O stock solution (see Recipes)
Staining solution for mineralized osteoblasts (see Recipes)

Equipment

Tissue culture set up
Pasteur pipette
Pasteur pipette tip
Light microscope
Pall Life Sciences acrodisc ayringe filter (0.2 μM HT Tuffryn membrane low-protein binding non-pyogenic) (Life Sciences, catalog number: PN 4192 )
0.2 μM nylon filter
24-well plates

Procedure

Plating cells for differentiation
1. Plate 50,000-60,000 osteosarcoma cells per well in 24-well plates in DMEM/10% FBS/ Penicillin-Streptomycin. This can be your cell line of interest too. Change medium according to your cell line of interest.
2. Next day or when they reach confluence (usually in 36 h), switch medium to differentiation medium. Usually cells will not differentiate unless they are at least 80-90% confluent. All differentiation medium is regular DMEM/10% FBS/PS supplemented with the differentiation cocktail indicated in steps 3 and 4.
3. For osteogenesis, use 10 mM beta-glycerol phosphate and 100 μg/ml ascorbic acid. These are the final concentrations.
4. For adipogenesis, use 10 μg/ml Insulin, 100 nM dexamethasone, 250 μM IBMX and 200 μM Indomethacim. These are the final concentrations. How to make stocks is described later.
5. Feed cells every 2-3 days with fresh differentiation medium made each time during feeding. While feeding make sure Pasteur pipette tip used for aspirating medium does not touch cell layer as cells attach loosely during differentiation, especially during adipogenesis.
6. Stain cells for osteogenesis and adipogenesis at 2, 5, 8, 10, 12 and 15 days.
Alkaline phosphatase assay to measure osteoblast differentiation.
This is a colorimetric assay where the yellow substrate. 4-Benzoylamino-2, 5-dimethoxybenzenediazonium chloride hemi (zinc chloride) salt, Azoic Diazo No. 24 is hydrolyzed by alkaline phosphatase to yield a purple dye precipitate.
Prepare fixation solution and staining solution before starting assay. Protect solutions from light fiter preparation.
1. Plate required cells in differentiation medium (usually 50,000 cells/well in 24-well plates).
2. Aspirate off medium from cells plated in differentiation medium at designated time.
3. Wash once with 1x PBS. Each washing step involved adding 1x PBS followed by aspiration.
4. Add 1 ml of fixation solution per well and incubate at RT for 1 min.
5. Wash wells with 1x PBS once.
6. Add I ml of staining solution per well.
7. Incubate in the dark for 30 min at RT and check color development. Development of purple-violet stained areas in well indicates positive reaction. If necessary, continue reaction for another 30 min at RT. Record increased incubation time. Do not exceed 1 h.
8. Stop reaction by washing quickly with dd water or tap water.
9. Do not forget to photograph plates before proceeding to Alizarin red S staining for mineralization.
Alizarin red S staining to detect mineralization
Alizarin red S is a calcium-sensing dye. Differentiated osteoblasts deposit large amounts of extracelluar calcium (a hallmark of mineralization) that can be detected by complexing with Alizarin red S. Calcium deposits appear as bright orange-red stained areas.
1. After photographing the plates stained for alkaline phosphatase, proceed to Alizarin red S staining by washing the stained plated twice with 1x PBS whose pH has been adjusted to 4.2 with 0.1% NH₄OH.
2. Aspirate the PBS and add enough Alizarin red S staining solution to cover the cellular monolayer. Incubate the plate at RT in the dark for 45 min.
3. Carefully aspirate the Alizarin red S solution and wash the plate fours time wits distilled water.
4. Extracellular calcium deposits appear as brightly stained orange-red areas.
Oil red O staining assay to measure adipogenic differentiation
This staining assay is based on the principle that the lipophilic dye Oil Red O is retained in fat globules of adipocytes that can be seen under a microscope.
1. Aspirate medium and wash cells once in PBS.
2. Fix cells in 4% PFA at RT for 15 min.
3. Wash twice with ddH₂O for 2 x 5 min.
4. Stain with Oil-Red-O working solution for 15 min at RT.
5. Wash with 1x PBS or ddH₂O for 3 x 5 min.
6. Rinse cells with 50% isopropanol once at RT.
7. Rinse cells with 1x PBS or ddH₂O.
8. The red stained lipid droplets can be visualized by light microscopy. The adipocytes are also readily visualized in normal microscopy through their rounded morphology and markedly enhanced light reflection of larger lipid droplets.
9. To count total cells, stain with Mayer's HemTox stain for 15 min at RT and wash 2x with dd water.

Recipes

Beta-glycerol phosphate can be prepared by making a 100x stock of 1 M solution. Pre-heat water for ease of dissolution. The formula weight of the compound depends on the degree of hydration and can be determined by using the Lot Number provided on the bottle. Filter using a Pall Life Sciences Acrodisc syringe filter. Make 1 ml aliquots and freeze in -20 °C.
1,000x ascorbic acid solution can be prepared by dissolving 1 gram of Ascorbic acid in 10 ml of ddw (double-distilled water). Filter using a Pall Life Sciences Acrodisc Syringe filter. Make 1 ml aliquots and freeze in -20 °C.
Dexamethasone (FW 392.5)
Dissolve 0.0196 g in 10 ml absolute ethanol to yield 5 mM stock; store at -80 °C. Prepare fresh working stock by diluting in 100% ethanol to 100 μM.
3-Isobutyl-1-methylxanthine (IBMX) (FW 222.2)
Dissolve 100 mg IBMX in 1.8 ml DMSO and sterile filter through 0.2 μM nylon filter to make 250 mM stock (1,000x). Aliquot and store at -20 °C or below.
Indomethacin (FW 357.8)
Dissolve 0.5367 g indomethacin in 7.5 ml DMSO, and filter through 0.2 μm nylon filter to sterilize for 200 mM (1,000x) stock. Dispense 1-2 ml aliquots in sterile cryovials, and store in opaque box to protect from light. Store at -70 °C or below.
Insulin, recombinant human
Dissolve 50 mg insulin in 10 ml EBSS acidified with 50 μl of 0.005 N HCl to yield 500x stock, and sterile filter. Do not forget to use low protein binding filters, as described in equipment. Dispense 1 ml aliquots in sterile cryovials and store at -70 °C or below for up to 2 years. Thawed, working stock can be stored at 4 °C for up to 1 month. Alternatively, sterile-filter acidified EBSS and use the sterile solution to make insulin under sterile conditions.
Fixation solution for osteogenesis
20 ml dd water + 30 ml acetone + 400 μl citrate concentrated solution.
Staining solution for osteogenesis
48 ml dd water + 2 ml Naphthol AS-MX phosphate alkaline solution, 0.25% + 1 complete capsule of fast blue RR salt
Oil-Red-O stock solution for staining adipocytes
Dissolve 500 mg of Oil-Red-O in 100 ml of isopropanol. For working stock, dilute stock as 2 parts dd water + 3 parts Oil Red O stock. Shake, Incubate at RT for 15 min and filter using a Pall Life Sciences Acrodisc Syringe filter. Use within 1 h of preparation.
Staining solution for mineralized osteoblasts
Dissolve 2 g Alizarin Red S in 100 ml distilled water, mix, and adjust pH to 4.2 with 0.1% NH₄OH to prepare the Alizarin Red S staining solution. Filter the dark-brown solution using a using a Pall Life Sciences Acrodisc Syringe filter. And store it in the dark in a foil-covered or amber colored bottle.
Note: The correct pH of the solution is critical. The solution is stable at RT for 2-3 weeks. If you plan on using it after a month, check pH to ensure pH is still 4.2.

Acknowledgments

This investigation was supported by PHS Grants AR051358 from the NIAMS and DE013745 from the NIDCR, and by an NCI UO1 award (to SHO). UBR is a recipient of a fellowship from The Children's Cancer Research Fund in memory of Dr A Rausen. AM is a recipient of a research grant from St Baldrick's Foundation. JAP is a postdoctoral fellow of the American Cancer Society. SHO is an Investigator of the Howard Hughes Medical Institute. This protocol is associated with the manuscript Basu-Roy et al. (2012).

References

Basu-Roy, U., Seo, E., Ramanathapuram, L., Rapp, T. B., Perry, J. A., Orkin, S. H., Mansukhani, A. and Basilico, C. (2012). Sox2 maintains self renewal of tumor-initiating cells in osteosarcomas. Oncogene 31(18): 2270-2282.