Thank You for sharing this protocol : I have 4 questions1- How...

1- How could I determine the PH in my reaction? I do the same volumes mentioned in the kit which differ according to the plate size (usually do 10 cm petri dish , without assessing the PH) I didn`t get proper greenish bluish staining , usually more brownish , I incubate them for 24 hours in dry 37 degree incubator without co2 as recommended.

If you are using a kit, i would strictly follow the kit instructions. I expect most reagents would be similar to our protocol. I have never seen any brownish staining in my experiments, it would be helpful to post your photo here.

2- My treatment is UV irradiation , do you have any idea how long should I keep cells after UV ttt to be able to attain senescence? in some paper they left them up to 12 days which is hard for my cells to be left all this time without need to be passaged?

I would not expect senescence to be induced right after UV irradiation. It would depend if the cells are able to repair the UV damage in the first place. If you predict to see replicative senescence, it is normal that it would take 12 days or more, or multiple passages until the cells are not able to replicate anymore. In our case, our manipulation permanently arrested cells at G1 phase, that's why it took only a few days to see the senescence phenotypes.

3- What is the recommended initial number or confluency of the flask prior to ttt if you intend to keep cells 5 days for ex. before fixation and staining?

It depends very much on the doubling time of the cells and how tolerance the cells are to seeding at very low confluence. If your cells are replicating for 5 days (without any growth arrest), then it is unlikely that the cells would be undergoing senescence in the first place. If your treatment is supposed to slow down growth but does not induce cell death, you could try to seed 10-15% of the growth surface area first.

4- I use the fixative solution that come with the kit and it is mentioned to be kept for 12-15 min. before washing, Is this different than paraformaldehyde 4% you are using?

Again, I am not familiar with the kit. However, PFA is one the most common fixatives. I would assume it could be the same or similar.