Aorta Single-Cell Isolation Protocol

Aortas were dissected from the aortic root to the top of the diaphragm after perfusion of the vasculature, and cleared of adventitial fat. Single cell suspensions were obtained by digesting the tissue for 1 h at 37°C in PBS with 450 U/ml collagenase type I, 125 U/ml collagenase Type XI, 60 U/ml hyaluronidase type I-S, and 60 U/ml DNase I (all from Sigma), as previously described [28] (link). Digests were filtered thru a 70 µm filter and resuspended in FACS buffer.

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Vu D.M., Tai A., Tatro J.B., Karas R.H., Huber B.T, & Beasley D. (2014). γδT Cells Are Prevalent in the Proximal Aorta and Drive Nascent Atherosclerotic Lesion Progression and Neutrophilia in Hypercholesterolemic Mice. PLoS ONE, 9(10), e109416.

Publication 2014

collagenase type I collagenase Type XI hyaluronidase type I-S DNase I

Adventitial Aortas Aortic root Buffer Cell Collagenase Diaphragm Dnase Hyaluronidase Perfusion Tissue

Corresponding Organization :

Other organizations : Tufts Medical Center, Tufts University

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Variable analysis

independent variables

Digestion time (1 h)
Digestion temperature (37°C)
Digestion solution composition (PBS with 450 U/ml collagenase type I, 125 U/ml collagenase Type XI, 60 U/ml hyaluronidase type I-S, and 60 U/ml DNase I)

dependent variables

Single cell suspensions obtained from aortic tissue

control variables

Aortic tissue source (aortic root to top of diaphragm)
Perfusion of vasculature
Removal of adventitial fat

controls

No positive or negative controls were explicitly mentioned.

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