BAEC serum starvation and permeability test - (May/21/2009 )
Hello all I appreciate your time in reading this and hopefully providing any feedback.
I am currently studying permeability in the endothelium of vessels. I have read a paper that has shown up regulation of a water channel protein, aquaporin-1, by a hormone arginine vasopressin on trophoblast placenta cells. I hope to achieve upregulation of aquaporin-1 by arginine vasopressin on bovine aortic endothelial cells. In the paper that I am basing my experiment on, placenta cells were grown with RPMI 1640 medium supplimented with 5% FBS & antibiotics on 6 well plates at 100k cells/well for 24 h. Cells were cultured in serum free and hormone deprived RPMI for another 12 h before treatments. Treatments consisted of 0.1nM and 10nM AVP formulations in RPMI medium with 1% FBS. Controls were incubated in medium containing 1% FBS alone.
The results of this paper showed a 5 fold increase in AQP-1 level with 0.1nM AVP.
My experiment is different because I grow my BAEC cells into a monolayer and then measure hydraulic conductivity in a cell chamber to quantify permeability. I have carried out the following experiments to understand starvation's effect on permeability and integrity of the monolayer.
Grow cells plated at 128k on 12 well dish with 10% FBS in MEM. Grow for 3-4 days.
1. 0% FBS in Minimum Essential Media
2. 1% FBS in MEM
3. 0% FBS in Optimem
1. 1% FBS in MEM with 0.1 nM AVP
2. 1% FBS in MEM
I ran 3 pairs, each being starved 1 of 3 ways and then ran treatments to test the effect of AVP. My results were not only much higher then my normal baseline (5.4E-6 cm/s) but also showed a weird trend.
Starvation technique and Hydraulic Conductivity
1. 16.86 E-6 cm/s for both control and treated
2. 17.90 E-6 cm/s control 14.11 E-6 cm/s treated
3. 18.92 E-6 cm/s control 13.10 E-6 cm/s treated
so the AVP decreased the Hydraulic conductivity and overall it looks like the starvation made the junctions very loose. I plan on running a test to see the effect of starvation alone. I would like any ideas that I am not considering to make this experiment work and keep the integrity of the mono layer. I would appreciate any help.
how would u think of growing big by taking just growth hormone and not having food.
never beleive totally in an article. try your own conditions.
tats what i noticed when i do expts.
when ur cells are starved they think primarily of surviving but not of hydrulic conductivity and when starved they dont have food to synthesise proteins(aquaporins). so it might show weird effects instead of the results thought.
although i never did the type of expt, i suppose u to check the hormones effect in normal media(10 % fbs) and varying hyraulic presures instead(we give anti oxidants when we give them oxidative stress and generally have not seen any good protective activity of those antioxidants in controls because they are already fine while the cells with stress get saved when supplemented with anti oxidants). for example higher hydraulic pressure may stimulate increase in aquaporin expression levels for dynamic transfer of liqids, but the time taken to express the increase may not b sufficient and cells may swell and die. by giving the hormone u may accelerate the proces(increase in no. of aquaporins) n save the cells.
generally to keep cells in static, living, non dividing condition we keep them in 3% fbs media for short timed tests. lower than 2% fbs will cause slow death of cells .
hope this helps