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 The samples were transferred to the laboratory environment for processing the same day as withdrawn. 1.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 …

Divulgazioni

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

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Citazione di questo articolo
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).

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