P3 hAMSCs were seeded in six-well plates at the density of 1 × 105 cells/cm2. When hAMSCs reached 60% confluence, cells were induced for osteogenic differentiation and chondrogenic differentiation, respectively. For osteogenic differentiation, hAMSCs were cultured with the osteogenic medium for 21 days. The osteogenic medium contained 10% fetal bovine serum (FBS), 1% penicillin/streptomycin, 1% L-glutamine, 1% nonessential amino acids, 50 μg/ml vitamin C, 10 mM β-glycerophosphate, and 10 nM dexamethasone (Solarbio, Beijing, China). Afterwards, the osteogenesis was detected by alzarin red staining (Solarbio, Beijing, China). For chondrogenic differentiation, hAMSCs were cultured with chondrogenic differentiation medium (Chondrogenic-Inducer Reagent; Cyagen) for 14 days and assessed by alcian blue staining (Solarbio, Beijing, China).
Alcian blue staining
Manufactured by Solarbio
Sourced in China, Germany
Alcian blue staining is a laboratory technique used to detect and visualize the presence of acidic polysaccharides, such as glycosaminoglycans, in biological samples. It is a commonly used staining method in histology and cytology.
Automatically generated - may contain errors
Lab products found in correlation
Oil red o staining, by Solarbio (4 mentions)
Alizarin red staining, by Merck Group (2 mentions)
Alizarin red staining, by Solarbio (2 mentions)
Dexamethasone (dex), by Solarbio (1 mentions)
Vitamin c, by Solarbio (1 mentions)
β glycerophosphate, by Solarbio (1 mentions)
Stro 1, by Proteintech (1 mentions)
Trypsin, by Solarbio (1 mentions)
Accuri c6 flow cytometer, by BD (1 mentions)
Chondrogenic differentiation basal medium, by Cyagen (1 mentions)
Bcip nbt alkaline phosphatase color development kit, by Beyotime (1 mentions)
Complete chondrogenic medium, by Cyagen (1 mentions)
Alizarin red solution, by Solarbio (1 mentions)
Adipogenic differentiation medium, by Cyagen (1 mentions)
Osteogenic differentiation medium, by Cyagen (1 mentions)
Alizarin red s staining, by Solarbio (1 mentions)
Alp staining, by CWBIO (1 mentions)
Alizarin red, by Solarbio (1 mentions)
H e staining, by Solarbio (1 mentions)
Masson trichrome staining, by Solarbio (1 mentions)
9 protocols using alcian blue staining
1
Osteogenic and Chondrogenic Differentiation of hAMSCs
2
Isolation and Characterization of PDLSCs
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This research was conducted in adherence with the Declaration of Helsinki. The ethical approval for this study was approved through the Medical Ethical Committee of the School of Stomatology, Shandong University (Protocol number: 20201206). The informed consent and the assent were taken from the participants as well as their guardians. The methods of the culture and identification of PDLSCs were in accordance with our previous research (Xi et al., 2021 (link)). The cultured PDLSCs were isolated from the human periodontal ligament (PDL) tissues in premolars from 12- to 16-year-old systemically healthy orthodontic individuals who undergone tooth extraction. 0.25% trypsin (Solarbio, Beijing, China) was applied to separate PDLSCs for passage. PDLSCs at passages 3–6 were utilized in the follow-up experiments.
The flow cytometric examinations of STRO-1, CD146, CD34, CD45 (Proteintech Group, Chicago, IL, United States) were carried out by using BD Accuri™ C6 flow cytometer (BD Biosciences, Milan, Italy) as described previously (Xi et al., 2021 (link)).
The multidirectional differentiation potential of PDLSCs was confirmed through alizarin red staining (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany), oil red O staining (Solarbio) as well as alcian blue staining (Solarbio) as described previously (Xi et al., 2021 (link)).
The flow cytometric examinations of STRO-1, CD146, CD34, CD45 (Proteintech Group, Chicago, IL, United States) were carried out by using BD Accuri™ C6 flow cytometer (BD Biosciences, Milan, Italy) as described previously (Xi et al., 2021 (link)).
The multidirectional differentiation potential of PDLSCs was confirmed through alizarin red staining (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany), oil red O staining (Solarbio) as well as alcian blue staining (Solarbio) as described previously (Xi et al., 2021 (link)).
3
Multilineage Differentiation Assay
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The method was consistent with our previous study.13 (link) PDLSCs were cultivated in the osteogenic, adipogenic or chondrogenic induction medium for three weeks. Then cells were performed by alizarin red staining (Sigma–Aldrich; Merck KGaA, Darmstadt, Germany), oil red O staining (Solarbio) and alcian blue staining (Solarbio), respectively.
4
Isolation and Differentiation of hAMSCs
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A schematic diagram of the experimental procedure is shown in Figure 1 . In this experiment, hAMSCs were isolated from the amnions of human placentas in accordance with a previous protocol, and relevant informed consent was provided by each donor before the operation.35 (link)-37 (link)
LG-DMEM/F12 medium containing 10% fetal bovine serum (FBS), 1% penicillin/streptomycin, 1% L-glutamine, and 1% nonessential amino acids was used to culture the hAMSCs. The morphology of the hAMSCs was observed under an inverted microscope after three days of cultivation. Third-generation (P3) hAMSCs were used in subsequent experiments. The stemness of the hAMSCs was tested by osteogenic and chondrogenic differentiation according to previous studies.35 (link)-38 (link)
Briefly, hAMSCs were seeded in a six-well plate, and the osteogenic medium (10% FBS, 50 mg/ml ascorbate, 100 nM dexamethasone, and 10 mM β-glycerophosphate) was added when the cell density reached 60%. The results were detected with the BCIP/NBT Alkaline Phosphatase Color Development Kit (Beyotime, China) and alizarin red staining (Solarbio, China). In terms of chondrogenic differentiation, we used Chondrogenic Differentiation Basal Medium (Cyagen, USA) to induce hAMSCs for 14 days; alcian blue staining was used for detection (Solarbio).
LG-DMEM/F12 medium containing 10% fetal bovine serum (FBS), 1% penicillin/streptomycin, 1% L-glutamine, and 1% nonessential amino acids was used to culture the hAMSCs. The morphology of the hAMSCs was observed under an inverted microscope after three days of cultivation. Third-generation (P3) hAMSCs were used in subsequent experiments. The stemness of the hAMSCs was tested by osteogenic and chondrogenic differentiation according to previous studies.35 (link)-38 (link)
Briefly, hAMSCs were seeded in a six-well plate, and the osteogenic medium (10% FBS, 50 mg/ml ascorbate, 100 nM dexamethasone, and 10 mM β-glycerophosphate) was added when the cell density reached 60%. The results were detected with the BCIP/NBT Alkaline Phosphatase Color Development Kit (Beyotime, China) and alizarin red staining (Solarbio, China). In terms of chondrogenic differentiation, we used Chondrogenic Differentiation Basal Medium (Cyagen, USA) to induce hAMSCs for 14 days; alcian blue staining was used for detection (Solarbio).
5
Multilineage Potential of HUCMSCs
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HUMSCs were assessed using Osteogenic-Differentiation Medium, Adipogenic-Differentiation Medium, and Complete Chondrogenic Medium (Cyagen Biosciences, Santa Clara, CA, USA). At the end of the incubation period, the differentiation of HUCMSCs was detected by three kinds of staining. Alizarin red solution (Solarbio, Beijing, China), oil red-O staining (Solarbio, Beijing, China), and alcian blue staining (Solarbio, Beijing, China) were used to detect osteogenic differentiation, lipogenic differentiation, and chondrogenic differentiation according to manufacturer’s instructions, respectively.
6
Tri-lineage Differentiation of MSCs
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Tri-lineage differentiation capacity of the indicated MSCs toward adipocytes, osteoblasts, and chondrocytes was verified, as we recently described, with several modifications1 (link),18 (link). In detail, 5 × 104 hESC-MSCs and BM-MSCs were seeded in 12-well plate wells in the aforementioned MSC culture medium for 2 to 3 days. Then, the medium was replaced with the commercial tri-lineage differentiation medium (adipogenic-, osteogenic-, and chondrogenic-differentiation; Stem Cell Technologies, USA), respectively. After a 2-week induction, the generated adipocytes, osteoblasts, and chondrocytes were dyed with Oil Red O staining (Solarbio, China), Alizarin Red S staining (Solarbio, China), and Alcian Blue staining (Solarbio, China), respectively.
7
Chondrocyte Differentiation Assays
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Human endplate cartilage cells were seeded in 24-well plates with a density of 1 × 105/ml. After the treatment was acquired, alkaline phosphatase (ALP) staining (CWBIO, China), Alcian blue staining (Solarbio, China), and Alizarin red (Solarbio, China) staining was performed according to the manufacturers’ instructions.
8
Histological and Immunohistochemical Analysis of Subcutaneous and Calvarial Tissue
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After μCT imaging, the retrieved subcutaneous masses and calvarial specimens were decalcified and subjected to paraffin embedding, followed by sectioning. The sections were deparaffinized and subjected to H & E staining (Solarbio, Cat# G1120), Masson trichrome staining (Solarbio, Cat#G1340), Modified Saffron-O and fast green stain kit (for bone; Solarbio, Cat#G1371) and Alcian blue staining (Solarbio, Cat# G2541) as described [[83] (link), [84] (link), [85] (link), [86] (link), [87] (link)]. For immunohistochemical (IHC) staining, the tissue sections were deparaffinized, rehydrated, antigen-retrieval treated, blocked and incubated overnight with primary antibody against Collagen II (1:200 dilution; Abcam; Cat# ab307674), followed by stained with biotin-labeled goat anti-rabbit IgG and horseradish peroxidase-conjugated-labeled streptavidin. The staining results were recorded under a bright field microscope.
9
Multilineage Differentiation of MSC-Seeded β-TCP Granules
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The MSC-seeded β-TCP granules from a patient with humerus non-union were cultured for 4 days, and cells were digested by pancreatin (Sigma) and suspended. Then, the cells were passaged, and the first-passage cells were collected.
For osteogenic induction, the cells were seeded into a six-well plate and cultured with osteogenic induction solution. At day 21, the cells were washed by PBS and fixed with 95% ethyl alcohol for 30 minutes. Then alizarin red staining (Solarbio, USA) was performed for 30 min at 37°C.
For adipogenic induction, the cells were seeded into a six-well plate and cultured with adipogenic induction solution A (Cyagen, USA) for 3 days, which was then changed to adipogenic induction solution B (Cyagen) for 1 day. At day 21, the cells were washed by PBS, fixed with 4% paraformaldehyde for 15 min, and then stained by oil red (Sigma).
Chondrogenic induction was performed as before19 . In brief, aliquots of 250,000 cells were suspended and centrifuged for 5 min at 600 g in a 15 mL tube. Then, 0.5 mL chondrogenic medium (Cyagen) was added into the tube. After 48 h incubation, the cell pellets were placed into the tube. Medium was changed twice a week. At 28 days, the pellets were fixed with 4% paraformaldehyde for 15 min, followed by alcian blue staining (Solarbio, China).
For osteogenic induction, the cells were seeded into a six-well plate and cultured with osteogenic induction solution. At day 21, the cells were washed by PBS and fixed with 95% ethyl alcohol for 30 minutes. Then alizarin red staining (Solarbio, USA) was performed for 30 min at 37°C.
For adipogenic induction, the cells were seeded into a six-well plate and cultured with adipogenic induction solution A (Cyagen, USA) for 3 days, which was then changed to adipogenic induction solution B (Cyagen) for 1 day. At day 21, the cells were washed by PBS, fixed with 4% paraformaldehyde for 15 min, and then stained by oil red (Sigma).
Chondrogenic induction was performed as before19 . In brief, aliquots of 250,000 cells were suspended and centrifuged for 5 min at 600 g in a 15 mL tube. Then, 0.5 mL chondrogenic medium (Cyagen) was added into the tube. After 48 h incubation, the cell pellets were placed into the tube. Medium was changed twice a week. At 28 days, the pellets were fixed with 4% paraformaldehyde for 15 min, followed by alcian blue staining (Solarbio, China).
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