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.
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
2. Isolation of valve cells
3. In vitro calcification assay
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 heart in the middle of the ventricles with scissors number 4. (D) Open the heart toward the aortic valve with scissors 3. Use the thin tweezers 7 and 8 to carefully dissect the aortic valve. The valve is visible and has some black dots that are characteristic of mice valve tissue (blue arrow). (E) Increase the magnification to better visualize the aortic valve. Isolate the valve with the small scissors 4; (F) maintain the tissue with tweezers 7.
Figure 2: Representative description of mouse valve cell isolation. Abbreviations: HEPES = 4-(2-hydroxyethyl)-1- piperazineethanesulfonic acid; RT = room temperature; DMEM = Dulbecco's modified Eagle medium; FBS = fetal bovine serum.
The authors have nothing to disclose.
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 |