3D Collagen Gel Contraction AssayListed By : Michelle Bendeck
University of Toronto
CategoryGenePathwayDiseaseMember Organization
Laboratory protocolRemodeling and fibrosisCardiovascularNAVBO
Collagen Gel Contraction Protocol


1. Culture Rat medial SMCs in DMEM supplemented with 10% FCS and 2% Pen/Strep

2. Prepare Collagen Gel as follows:

1.5 mg/ml Collagen Solution: (8 ml)

Mix:
4.0 ml Vitrogen collagen (3 mg/ml stock)
0.5 ml 0.01M NaOH
3.5 ml DMEM/FCS


For 3.2mg/ml stock use:
3.75 ml Col I
0.47 ml 0.01 M NaOH
3.8 ml DMEM

KEEP COLD IN ICE WATER !!!

4. Seed cells within collagen gel

• Trypsinize cells
• Resuspend in Phenol Red Free Media (w/ serum) and Count using Trypan Blue method
• Remove aliquot containing 3.6 x10^6 cells
• Dliute Aliquot to 4.0 ml and mix with collagen solution
• Add 0.5 ml aliquots of cold solution to 24 well plates

Perform all experiments in Triplicate

Final concentrations: 3x10^5 cells / ml : 1.5x10^5 cells/well

5. Polymerize collagen by Incubating @ 37 C for 45 min in TC Incubator

6. Cut gels from sides of well with scalpel and detach from bottom by pipetting 1 ml media (10% FCS, 2% PS +/- 100, 200, 300, 400, 500 ug/ml Doxycycline) under gel.

7. Incubate @ 37 C and photograph wells @ 2, 4, 6, 12, 24, 48, 72 hrs

8. Measure area of gel at each time-point and express as % contraction of original gel area (t = 0)
can also use diameters

Collagen Gel Contraction Protocol

1. Prepare Fresh Phenol Red Free Media

2. Trypsinize Cells and Count

3. Prepare Appropriate Number of Cells at 3.0x10^5 cells/ml in 4 ml Media

4. Prepare Collagen Solution without NaOH (KEEP IN ICE WATER)

5. Remove Collagen Solution from Ice water and quickly add 0.01M NaOH Let Stand at r.t. one minute

6. Add cell solution and mix thoroughly

7. Pipette 0.5 ml gel solution into wells of a 24 well plate (assay in triplicate)

8. Incubate at 37 C for 1H to polymerize Gels

9. Prepare Doxycycline Treated media using 10 mg/ml Dox stock (in Ca2+ and Mg2+ free PBS) and dilute into Phenol Red Free Media (Media will go cloudy at high concentrations of DOX)

10. Remove Polymerized Gels from incubator and GENTLY detach gels using a sterile spatula (twice around gel circumference should detach gel)

11. GENTLY pipette 1 ml of Doxy Treated or Control Media into each well. This should gently lift the gels off the bottom of the well. GELS SHOULD BE FLOATING

12. Incubate Gels for necessary timepoints at 37 C in T.C. Incubator

13. Image Gels with Nikon Digital Camera at necessary timepoints or remove conditioned media samples.

14. Once imaged, Gels can be fixed for 1h in 4% paraformaldehyde and stored at 4 C in PBS for further analysis
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