Alcian Blue Staining to Visualize Proteoglycans: A Technique to Detect Proteoglycans in Mesenchymal Stem Cell Differentiated Chondrocytes Culture In Vitro
Published: April 30, 2023
Abstract
Source: Schlaefli, P. et al., An Enzymatic Method to Rescue Mesenchymal Stem Cells from Clotted Bone Marrow Samples. J. Vis. Exp. (2015).
This video describes the alcian blue staining procedure for locating proteoglycans secreted by functional chondrocytes. This method helps determine the success of in vitro differentiation of mesenchymal stem cells into mature chondrocytes.
Protocol
NOTE: Canine bone marrow aspirates from the iliac crest were collected with dog owner's consent. Canine (approx. 10 ml) bone marrow aspirates were anti-coagulated by the addition of 15 ml of 3.8% sodium citrate immediately after withdrawal in the operation theatre. The samples were transferred to the laboratory environment for processing the same day as withdrawn. 1. Preparation of Urokinase (Prior to 1st Use) Reconstitute urokinase …
Disclosures
The authors have nothing to disclose.
Materials
| Basal Medium Components | |||
| PenStrep 100X | Gibco | 15140122 | |
| Human FGF-basic | Peprotech | 100-18B | |
| MEM Alpha w/ Nucleoside, w/stable Glutamine | Amimed | 1-23S50-I | |
| FBS Heat Inactivated | Amimed | 2-01F36-I | |
| Amphotericin B | Applichem | A1907 | |
| Adipogenic Medium Components | |||
| DMEM-HAM F12 + GlutaMAX | Amimed | 1-26F09-I | |
| Chondrogenic Medium Components | |||
| Biopad – sponge shaped medical device | Euroresearch | ||
| L-proline | Sigma | P5607 | |
| Insulin-Transferrin-Selenium X | Gibco | 51500056 | |
| Human transforming growth factor-β1 | Peprotech | 100-21 | |
| Alcian Blue 8GX | Sigma | A3157 | |
| Nuclear fast red | Sigma | N8002 | |
| Generic Tri-Sodium citrate dihydrate | Applichem | A3901 | |
| PBS | Applichem | 964.91 | |
| Sulfuric acid (H2SO4) | Applichem | A0655 | |
| Dimethyl sulfoxide (DMSO) | Applichem | A1584 | |
| Magnesium chloride (MgCl2) | Applichem | A3618 | |
| Guanidine hydrochloride | Applichem | A1499 | |
| Consumables | |||
| 50 ml reaction tube | Axygen | SCT-50ML-25-S | |
| 10 ml syringe | Braun | 4606108V | |
| Sterican needle (22G) | Braun | 4657624 | |
| 1.7 ml Microtubes | Brunschwig | MCT-175-C | |
| 100 μm cell strainer | Falcon | 6.05935 | |
| sterile forceps | Bastos Viegas, SA | 489-001 | |
| sterile scalpel | Braun | 5518059 | |
| Primaria cell cuture dish | Falcon | 353803 | |
| C-Chip Neubauer Improved | Bioswisstech | 505050 | |
| cell culture flask – Flask T300 | TPP | 90301 | |
| Equipment | |||
| Microbiological biosafety cabinet class II | Skan | 82011500 | |
| water bath | Memmert | 1305.0377 | |
| Stripettes Serological Pipette 5ml | Corning | 4487-200ea | |
| microscope | Olympus | CKX41 | |
| humidified incubator Heracells 240 | Thermo scientific | 51026331 | |
| Heraeus Multifuge 1S-R | Thermo scientific | 75004331 |
Tags
Cite This Article
Alcian Blue Staining to Visualize Proteoglycans: A Technique to Detect Proteoglycans in Mesenchymal Stem Cell Differentiated Chondrocytes Culture In Vitro. J. Vis. Exp. (Pending Publication), e21051, doi: (2023).