Ahmad Sidiqi
June 5th 2015
HRGEC in vitro Network Formation Assay in Matrigel
Preparation of HRGECs
1) Grow passage 2-6 of HRGECs (Sciencell, catalogue # 4000) on 1mg/ml fibronectin coated plates
– until 80-90 % confluency. Raise them in Endothelial Cell Medium (ECM – Sciencell, catalogue #
1001)
a. T25 flask per condition = 5 x 105 cells
b. T75 flasks per condition = 2-4 x 106 cells
2) Serum starve the cells for 3 hours by washing with PBS and raising cells in serum-reduced ECM.
Meanwhile…
Preparation of Matrigel aliquots
1) Pre-cool 1000 µL pipette tips and 1.5 mL tubes
2) Take Matrigel stock solution (Corning, New York, New York, catalogue # 354320) and place on
ice overnight - and make 500 – 1000 L aliquots (usable for 10 – 20 wells) in sterile 1.5 mL tubes
for storage at -20C.
Preparation of supplies
1) Pre-cool the following equipment
a. 200 L pipette tips – place these in the fridge
b. 96 well plate – place these in the fridge
2) Thaw Matrigel aliquots on ice overnight – place the ice box in the cold room or in fridge
3) Prepare VEGF working stock solution
a. Dilute stock to get 5 ng/uL
i. Divide into working stock aliquots (40 uL) and freeze down (-20C)
Plating of Matrigel and cells
1) Add 50 L cooled growth factor reduced Matrigel into wells using cool pipette tips (try to
minimize air bubbles – this can be done by avoiding pushing the residual volume in the pipette
tip).
2) Leave for a minimum of 30 mins at 37C (can leave longer if necessary – not longer than 2
hours).
3) While Matrigel polymerizing:
a. prepare ECM with or without VEGF (100ng/mL per condition )
4) Trypsinize HRGECs (usually 1 T75 or 100mm plate flask gives 2-4 million cells)
a. 2 mL trypsin x 3 – 5 mins at 37C
b. 2 ml HRGEC growth medium to inactivate trypsin
5)
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c. Take 10 L aliquot for counting with hemacytometer
Determine how many wells can be supplied with # of cells that you have (8-104cells per well)
Split cell suspension into separate aliquots for each condition
Spin the separate aliquots down (1500 rpm, 5 mins, RT)
Resuspend in appropriate pre-prepared media
Plate the endothelial cell mixture on top of Matrigel
Incubate for 6 hours at 37C
Imaging
1) Take pictures immediately after 6 hours using cell culture microscope and a USB.
2) 5 images per well – take one directly in the centre of the well, the top, the right side (from
centre), the left side (from centre), and the bottom of the well.
Analysis of networks
1) Measure the length of the networks from stalk to the edge of the tip for each of the 5 images,
per well, using Fiji (ImageJ) line measure tool
2) Add the total length of all 5 images together to get the total network length per experimental
well.
NOTES:
1) % of serum used is important
a. too much serum can lead to network formation without VEGF
b. too little serum can lead to NO network formation even with VEGF
c. in past:
i. 10% FBS too much (tubes even with no VEGF) – done July 17 2012
ii. 5% FBS too much (tubes even with no VEGF) – done Aug 3 2012
iii. 1% FBS too little (no tubes) – done Aug 13 2012
iv. Use 5% FBS – the amount of FBS found in stock ECM – Confirmed by Ahmad
d. literature:
i. 5% FBS 1
2) # of cells used is important
a. 1.2 x 104 cells/well for 96 well plate 1
b. 1 x 104 cells/well for 96 well plate 2
c. Use between 8-104 cells per 96 well plate (maximum) – Confirmed by Ahmad
3) some Matrigel lots can induce tube formation even in absence of serum 1. Its important to try
and use the same lot for each experiment. Make not of the lot number and if more Matrigel
needs to be purchased, try and purchase the same lot (not always possible as batches are sold
out or destroyed after some time by the company)
4) Timing between trypsinizing and plating the cells is also important. Taking too long in this step
will prevent the cells from forming networks, even in the presence of VEGF.
1. Ponce, ML: Tube formation: an in vitro matrigel angiogenesis assay. Methods in molecular biology
467: 183-188, 2009.
2. Szpaderska, AM & DiPietro, LA: In vitro matrigel angiogenesis model. Methods in molecular medicine
78: 311-315, 2003.
i.