Dear Jia,

I can see different ways to improve your current protocol:

1) HUVEC need to be totally confluent in order to perform shear stress experiments. We recently published different works on the existence of a fluid shear stressmechanosensory  junctional complex. In absence of junctions, this complex may not perform optimally.

2) When you culture HUVEC, do you add ECGS and heparin, in addition to 20% serum? Without those factors, HUVEC are not really happy. You also need to use them between P2 and P6. Senescent cells do not respond to flow and alter the ability of surrounding cells to align. I usually starve my cells with culture media containing lower serum concentrations to perform alignment experiments.

3) I recently publihed a paper on the existence of a shear stress set point: endothelial cells only align within a narrow range of shear stress magnitude. It could be possible that the magnitude you apply to your cells is either too low or too high. Testing different magnitudes might fix the problem.

4) While Ibidi chambers are appropriate for specific applications like live imaging, I prefer to use parallel plate chambers, it is more easy to control cell density and flow parameters with this system. 

Best,

Nico

Thanks Nico, I have actually been reading your papers in PNAS and eLIFE, and trying to follow your protocol. Are you still with Martin? We have a few collaborations with him on the shear stress projects.

To respond to your suggestions:

1, I have tried different densities, but never tried totally confluent cells. I will give it a try as you suggested.

2,I have cultured HUVEC(P2) in complete medium containing 20% serum and ECGs and Heparin, and used the same medium for the following flow experiment. After 72hr, I did see a few senescent cells. I noticed in your 2014 PNAS paper, you starved cells with 5% FBS medium for minimum 4 hrs prior to shear stress treatment. Did you then put HUVEC in complete medium under shear stress? 

3, so far I have only tried 15 and 20 dyn/cm2( I noticed you used 12 or 15 dyn/cm2). There was not much difference between these two. However they were all under 60-80% confluence.

4, Unfortunately we only have ibidi system in our lab.

If you don't mind, may I have your email address for the future questions?

Thanks again.

Jia

Hello Jia!

Indeed, I think I met your supervisor at a Gordon conference in January, it was interesting!

II really think that confluent cells would help a lot. Also, I always starve my cells and then shear them with the starvation medium. For the record, I always cut custom made tissue culture plastic slides. cells usually detach when seeded on glass slides but this should not be an issue with ibdi chambers. How long after seeding do you shear them? Do you coat the chamber with fibronectin or something similar? This might help, too.

Concerning the shear stress magnitude, I would tweak a little bit the system... the Ibidi chamber is very small so little changes in perfusion are going to have huge consequences on the magnitude (remember, a difference of 2-3 dynes/cm2 is enough to totally change the response). Let me know if you ever consider building a parallel plate system, i would be happy to provide the blueprints for the chambers and an order list.

You can directly contact me at the following adress: [email protected]

Cheers,

Nico

Hi Nico,

Thanks a lot. 

I normally started flow 24hr after seeding cells. The ibidi slides were coated with Fibronectin.

One more quick question: the starvation medium also contains ECGs and heparin?

Best regards

Jia

You are welcome!

For the starvation media, I only use FBS and antibiotics diluted in M199. other people in the lab dilute the complete medium. We see some differences between the different media but this is not drastic.

Nico

Thanks Nico. I will give it a try and come back to you once I get the result.

Best regards

Jia