Hi

I am doing the research linking autophagy and EMT, the factor is HBV infection.

Now I am doing a experiment like this: first transfected Huh751 cells with HBX, 48 hrs later, seeding in transwell system.

This is overivew of my protocol:

1, D0---seeding 3X10e5/3 ml each well of H751 cells

2, D1---transfection (DNA 3.3 ug/well, ration=4:1, using Fugene)

3, D3---trypsin cells, counting, re-seeding in 24-well transwell system.

200 ul upper chamber (5X10e5/ml). Meanwhile, I will add autophagy stimulator or inhibitor such as 3-MA, Rapa into upper chamber.

4, 6 hrs later, cristal violet stain, count and take picture.

The question is as following:

1) Is the protocol good?

2) The volumn: I am planning to add 200 ul in upper chamber, and the lower chamber, 750 ul.

3) cells setting: originally I seeded 3 cells, made 3 transfection---vector, HBX or HBV 1.2 plasmid, when coming to the transwell system, the setting would be SFM(upper chamber) /comp(lower compartment, means complete medium), Vector/SFM, Vector/comp,HBX/SFM, HBX/comp, HBX+3-MA/comp, HBV/SFM, HBV/comp, HBV+3-MA/comp.

Is this setting good?

Do I have a group of cells which would have SFM in the lower compartment as negative control, for each plasmid?

thx