Alcian Blue Staining to Visualize Proteoglycans: A Technique to Detect Proteoglycans in Mesenchymal Stem Cell Differentiated Chondrocytes Culture In Vitro

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 The samples were transferred to the laboratory environment for processing the same day as withdrawn.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 1^st Use)

1. Reconstitute urokinase using sterile phosphate-buffered saline (PBS) according to the manufacturer's instructions: Add 10 ml PBS to the septum-inlet flask using a 10 ml syringe.....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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