Immortalized Mouse Endothelial Cell Culture (Version 1.0)
Version
1.0
Notice
This page is the corresponding protocol tomestone page generated as part of the ATLAS-D2K shutdown in July 2025. Many links on this page may be broken.
Authors
Racheal Jappert; Jeffrey Pippin; Ying Zheng; Stuart Shankland
Keywords
[‘mouse’, ‘human’, ‘Adult vascular endothelium’]
Subjects
[‘Cell culture’, ‘Tissue culture’]
Release Date
2017-05-26
Abstract
Describes the procedure for maintaining mouse vascular endothelial cells (isolated from a defined source) in culture.
Introduction
YFP immortalized mouse endothelial cells have been isolated from endothelial reporter mice (Flk1::H2B-EYFP) crossed with ImmortoMice (containing the interferon driven thermo-sensitive T antigen from SV40). The cells were isolated via fluorescent assisted cell sorted for YFP expressing cells from full kidney liberase digestions.
Reagents
Growth Media
Mouse endothelial growth media is ordered from Cell Biologics catalog number M1168
- 500mL Endothelial Cell Media
- 0.5mL Hydrocortizone
- 5mL Antimycotic
- 0.5mL Endothelial cell growth supplement (ECGS)
- 5mL L-glutamine
- 0.5mL heprin
- 100 mL FBS
Note: Endo media kit comes with VEGF but do not add to the media. Kit comes with only 25mL FBS.
Procedure
Gelatin Coating Plates
Prepare fresh plates before use. DO NOT use older gelatin plates as the endothelial cells will not lie down as well.
1) Dissolve 100mg gelatin in 100 mL sterile water 2) Heat and thoroughly mix gelatin solution (solution can be stored at 4°C) 3) Run solution through steri-filter 4) Add 2.5 – 3 mL gelatin to 100mm Primaria dish and chill at 4°C 2-24 hours 5) Remove extra gelatin by aspiration 6) Immediately culture cells onto dishes
Growth Permissive Conditions:
1) Use freshly made gelatin coated plates on Primaria plastic 2) Add 50 units/mL interferon-ϒ (5ul per 10mL media) 3) Grow at 33°C and passage upon confluence (DO NOT allow to overgrow)
Growth Restrictive Conditions:
1) Use freshly made gelatin coated plates on Primaria plastic 2) No interferon-ϒ 3) Grow at 37°C and passage again if they become too confluent
What you should expect to see:
Endothelial cells can take up to 4 days to lie down completely; however, when using fresh gelatin plates, this usually happens in the first 24 hours. It is best not to change the media for the first 4 days after passaging so cells are not lost.
In growth permissive conditions, the endothelial cells will grow in colony-like clusters and should be passaged when the clusters are confluent, even if the plate as a whole is not confluent.
In growth restrictive conditions, cells will increase in size and expand out and may occasionally grow in vessel-like patterns. Cells do not typically grow in colony-like clusters once they have been thermo-shifted into restrictive conditions. Cells can be re-plated on day 3 or 4 of growth restriction for experiments.
Things you should watch for:
In growth permissive conditions, cells are prone to spontaneous transformation, usually occurring when cells are grown at a high density. Do not allow cells to overgrow or over-plate cells in order to prevent spontaneous transformation.
Ensure cells are dispersed and no clumps are present during passaging as it can introduce highly confluent foci that could transform. If clumps are an issue, use a cell strainer.
Use 50units/mL of INF, as lower doses can lead to selection of cell populations that do not require INF to proliferate. These populations will eventually dominate and will grow uncontrollably and cannot be transferred to growth restrictive conditions.
Expand and freeze down many stock vials of lower passage cells upon arrival. If cultures become dominated by spontaneously transformed populations, discard the cultures and revert back to your stock supply.
Monitor the temperatures of your incubators periodically with a thermometer to make certain that temperatures are correct for growth permissive and growth restrictive conditions.
Consortium
(Re)Building a Kidney (RBK) Consortium
