In order to test the possible involvement of irisin in inducing the production of calcium-rich deposits in vitro, ARS was carried out on DBSCs differentiated in osteogenic medium for 21 days. Once the culture medium was removed cells were rinsed with PBS and fixed in 10% formalin at room temperature for 10 min. Then, cells were washed with deionized water and stained with a 1% ARS solution (Alizarin Red powder was purchased from Sigma Aldrich, Milan, Italy) for 10 min at room temperature. The ARS solution was discarded at the end of the incubation time, and cells were rinsed twice with deionized water and then air dried.
Alizarin red powder
Manufactured by Merck Group
Sourced in Italy
Alizarin Red powder is a chemical compound used as a histological stain. It is a bright red crystalline powder that is soluble in water and alcohol. The primary function of Alizarin Red powder is to stain calcium deposits in biological tissues, allowing for the visualization of mineralized structures during microscopic analysis.
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Lab products found in correlation
Ascorbic acid, by Merck Group (2 mentions)
P nitrophenyl phosphate, by Merck Group (1 mentions)
2 2 azobisisobutyronitrile, by Merck Group (1 mentions)
3 4 5 dimethylthiazol 2 yl 2 5 diphenyltetrazolium bromide mtt, by Merck Group (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Gelatin, by Merck Group (1 mentions)
N isopropylacrylamide, by Merck Group (1 mentions)
N hydroxysuccinimide, by Merck Group (1 mentions)
1 ethyl 3 3 dimethylaminopropyl carbodiimide, by Merck Group (1 mentions)
Genipin, by Merck Group (1 mentions)
Methyl thiazolyl tetrazolium (mtt), by Merck Group (1 mentions)
β glicerophosphate, by Merck Group (1 mentions)
Alkaline phosphatase kit, by Merck Group (1 mentions)
Dexamethasone (dex), by Merck Group (1 mentions)
β glycerophosphate, by Merck Group (1 mentions)
Anti opn, by Abcam (1 mentions)
7 protocols using alizarin red powder
1
In Vitro Alizarin Red Staining
2
Chitosan-Based Hydrogel for Cell Culture
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Medium molecular weight CS (degree of deacetylation= 75–85%), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), N-hydroxysuccinimide (NHS), 2, 2'-azobis (isobutyronitrile) (AIBN), N-isopropylacrylamide (NIPAAm, 99%), genipin, p-nitrophenyl phosphate, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and alizarin red powder were supplied by Sigma-Aldrich (St. Louis, Missouri United States). 3-Mercaptopropionic acid (MPA), isopropyl alcohol, and gelatin were purchased from Merck. Dulbecco’s modified eagle’s medium F-12 (F-12 DMEM), fetal bovine serum (FBS), and antibiotics (Penicillin, Streptomycin), and all cell culture components were purchased from Invitrogen (Karlsruhe, Germany).
3
Resveratrol and Polydatin for Osteogenic Differentiation
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Antibodies were from Abnova. Ascorbic acid, β-glicerophosphate, Alizarin red powder, alkaline phosphatase staining kit, MTT, were from Sigma Aldrich. Resveratrol and Polydatin extracted from Polygonum Cuspidatum, according to the procedure described in the Patent EP1292320B1, were provided by Prof. Ravagnan. Resveratrol and polydatin were dissolved in ethanol at 100mM stock solutions 39 (link). Intermediate 1mM and 0.1mM stock solutions were freshly prepared by diluting in ethanol, 100mM stocks, 1:100 and 1:1000 respectively. Intermediate 1mM and 0.1mM stock solutions were diluted 1:1000 in culture medium just prior the use, to reach the final concentrations of 1.0µM and 0.1 µM. CTR samples carried 1:1000 dilution of vehicle. Control without vehicle didn't show any difference compared to control with vehicle. For this reason, the control without vehicle was not included in the data for simpleness.
4
Alizarin Red Staining of Mineralized hNPCs
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First, hNPCs were washed three times with PBS. Then, the cells were fixed with 4% formalin (#252549‐1 L, Sigma‐Aldrich) and then incubated overnight at 4°C. Afterwards, cells were rinsed twice with distilled water and 2% Alizarin red solution was added. After an incubation time of 45 minutes in the dark at room temperature, the cells were rinsed four times with distilled water and finally covered with PBS. The Alizarin red staining consisted of 2% Alizarin red powder (#A5533‐25G; Sigma‐Aldrich) dissolved into distilled water.
5
Alizarin Red Staining for Extracellular Calcium Deposition
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The extracellular calcium deposition was stained via alizarin red staining protocol (31 (link), 32 (link)). The medium was removed, and cells were washed with PBS three times and fixed with 4% paraformaldehyde. Subsequently cells were washed with PBS 3 times and stained in filtered 40 mM alizarin red (pH ~ 4.2) for 15 minutes in room temperature (Alizarin Red powder, Cas Number 130-22-3 Sigma). Finally, cells were washed, and the wells photographed in stereomicroscope (SMZ25) to identify calcified nodules in bright- orange- red color. For numerical quantification (33 (link)), stained cells were dissolved in 10% acetic acid. The suspended samples were heated to 80°c for 10 minutes. Then, samples were cooled on ice for 5 minutes, and centrifuged at 20,000 g for 15 minutes. Supernatant pH was adjusted to 4.2 and absorbance was measured in 405 nM.
6
Osteoblast Differentiation Assay
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1α,25-Dihydroxyvitamin D3, ascorbic acid, β-glycerophosphate, dexamethasone, Alizarin Red powder, and alkaline phosphatase kit were from Sigma Aldrich, Milan, Italy.
Anti-RUNX2 antibody was from Abnova, anti-Coll I, anti-OPN, and anti-BSP II were from Abcam, Cambridge, UK.
Anti-RUNX2 antibody was from Abnova, anti-Coll I, anti-OPN, and anti-BSP II were from Abcam, Cambridge, UK.
7
Alizarin Red Staining for Mineralization
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Alizarin Red powder (Sigma-Aldrich) was dissolved in deionized water to obtain 2% (w/v) solution; its pH was adjusted within a range of 4.1-4.3; and the solution was filtered. Samples were fixed in 4% paraformaldehyde and washed with DPBS before adding the Alizarin Red staining. The samples were incubated in the dark for 45 min. The staining was aspirated and samples were washed multiple times with deionized water until no reddish color of the water was observed. Following this step, the deionized water was added again and the samples were gently shaken overnight on an orbital shaker. This step was incorporated into the protocol due to the adhesive nature of Alizarin Red to remove any nonspecific binding of the staining to the samples.
Ca-to-P molar ratios were calculated based on the atomic concentration (at%) of Ca and P detected on the survey scans obtained from XPS analysis of the D21 biological samples (n = 2 per surface finish). High-resolution spectra of Ca2s and P2s were recorded.
Ca-to-P molar ratios were calculated based on the atomic concentration (at%) of Ca and P detected on the survey scans obtained from XPS analysis of the D21 biological samples (n = 2 per surface finish). High-resolution spectra of Ca2s and P2s were recorded.
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