Calcification Assay to Measure Calcium Concentration in Aortic Valve Interstitial Cells

In This Article

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. Prepare an aliquot of 50 mL of 1x phosphate buffered saline (PBS). Keep the solutions on ice.
Prepare 1 mg/mL and 4.5 mg/mL collagenase solutions an....

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Results

Surgical dissection series; tools shown; orange tissue under manipulation; close-up examination.
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 .......