Calcification Assay to Measure Calcium Concentration in Aortic Valve Interstitial Cells
Published: April 30, 2023
Abstract
Source: Bouchareb, R. et al., Isolation of Mouse Interstitial Valve Cells to Study the Calcification of the Aortic Valve In Vitro. J. Vis. Exp. (2021).
This video demonstrates an assay technique for assessing the calcification of the aortic valve interstitial cells in vitro. The assay helps to quantify the calcium concentration using the arsenazo III reagent, a calcium-sensitive dye that reacts with calcium ions to form a purple-colored calcium-arsenazo complex.
Protocol
All animal procedures described here have been approved by Icahn School of Medicine at Mount Sinai institutional core and use committee. 1. Preparation before valve cell isolation from adult mice Clean and sterilize all the surgical instruments shown in Figure 1A by using 70% v/v ethanol and subsequently autoclaving them for 30 min. clean the surgical workspace with 70% ethanol. Add 500 µL of penicillin-streptomycin to 50 mL of 10 mm HEPES. P…
Representative Results
Figure 1: Description of valve dissection. (A) Representative image of all the surgical instruments needed for the dissection, scissors 2 is needed to open the skin of the mouse and scissors 3 to open the chest. Tweezers 5 and 6 are needed to hold the skin and open the chest. (B) Leave 3 mm of tissue from the aorta (black arrow). (C) Cut the hear…
Declarações
The authors have nothing to disclose.
Materials
| 3 mm cutting edge scissors | F.S.T | 15000-00 | |
| Arsenazo-III reagent set | POINT SCIENTIFIC | C7529-500 | A kit to measure the concentration of calcium |
| Bonn Scissors | F.S.T | 14184-09 | |
| Calcium hydroxide | SIGMA -Aldrich | 31219 | |
| Collagenase type I (125 units/mg) | Thermofisher Scientific | 17018029 | |
| DMEM | Tthermofisher | 11965092 | |
| Extra fine graefe forceps | F.S.T | 11150-10 | |
| FBS | Gibco | 16000044 | |
| Fine forceps | F.S.T Dumont | ||
| HCl | SIGMA-ALDRICH | H1758 | |
| HEPES 1 M solution | STEMCELLS TECHNOLOGIES | ||
| L-Glutamine 100x | Thermofisher Scientific | A2916801 | |
| PBS 10x | SIGMA-ALDRICH | ||
| Penicillin streptomycin 100x | Thermofisher Scientific | 10378016 | |
| Sodium Pyruvate 100 mM | Thermofisher Scientific | 11360070 | |
| Standard pattern forceps | F.S.T | 11000-12 | |
| Surgical Scissors – Sharp-Blunt | F.S.T | 14008-14 | |
| Trypsin 0.05% | Thermofisher Scientific | 25300054 |
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Calcification Assay to Measure Calcium Concentration in Aortic Valve Interstitial Cells. J. Vis. Exp. (Pending Publication), e21280, doi: (2023).