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Isolation of endothelial cells, smooth muscle cells and fibroblasts.
The protocol is described briefly.
Requirements:
Endothelial Cell Medium : DMEM 500ml +50 ml FCS +5 ml P/S+ Supplement Pack without Amphotericin and Gentamycin.
Smooth Muscle Cell Medium with supplementary Packet
Dulbecco's Modification of Eagle's Medium (DMEM),
Stop medium : M 199+ 100 Ml FCS +5 ml PS
DMEM with 10 % FCS was used instead of Stop medium
Procedure
1. Preparation of T Flask coated with Gelatin
0.1 % Gelatin Sigma (G-2500) was prepared by dissolving gelatin in Sterile Deionised Water.
2. Preparation of Collagenase A :
0.01 g of Collagenase A ( Roche, Cat No: 103578) was taken and dissolved in 10 ml PBS and was passed through a sterile filter.
A.Isolation of Endothelial Cell :
An artery was taken and washed thoroughly outside and inside with PBS. The artery was then clamped with Arterial Clamps. Collagenase was injected in the artery with a thin needled syringe till the artery was totally filled. The artery was incubated in PBS at 37° C for 30 minutes. The artery was taken out and the arterial clamps was removed and the fluid from the lumen was collected in a tube, which was then filled with 5 ml Stop Medium. The tube was centrifuged at 1200 rpm for 10 minutes to sediment the cells from the lumen. The supernatant liquid was removed and the cell pellet was resuspended in a 25 ml culture flask previously coated with gelatin. 10 ml EC media was pipetted in the flask to support the growth of the Cell. The Flask was incubated at 37 °C.
