Hello Ankit, I am not sure about pH, maybe it also depends on a cell type you are working with. What kind of cells are you using? And how many cells do you use for isolation? I can recommend you a paper from colleagues, they isolated mitochondria from C2C12 cells, protocol is simple and easily reproducible - Elkalaf et al, 2013, in PLoS ONE. Good luck!

In the cytoplasm the pH is 7.2. Therefore during isolation it is nesessary to maintain this pH. The best buffer is MOPS. The buffer capacity of Tris buffer is much lower at this pH. In addition, Tris may damage combination pH electrodes. If pH is too acids (6-6.5) ot too alkaline (around 8) this may damage mitochondria. The detals are described in my book in my RG account.

What system are you working in?

Also, are you completely sure you are getting efficient lysis, and not just spinning down mainly whole cells?

Dr. Jakub Ziak correctly indicated that not only pH but other factors might affect the yield of mitochondria from the cultured cells. From my experience, these factors are as follows:

1. Ankit Sabharwal mentioned that the first centrifugation was performed at 600 g. In most cultured cells the mitochondria form a kind of ring or even a network. So, they are huge and thus a lot of mitochondria could be lost even at 600 g.

2. However, of more importance might be the fact that most researchers cultivate cells in the presence of antibiotics, usually PenStrep, It is well known that aminoglycoside antibiotics are mitotoxic and render the mitochondria inability to consume oxygen and after a number of passages the number of mitochondria per cell declines significantly.

3. Lysis of cells might damage mitochondrial membrane and thus they will not sediment at 11000 g., but at 100000 g. If you try this. In my experiments, I used hypotonic swelling method, which is described in my book "Practical mitochondriology". You can find this book at my page of Research gate. The book is also available at Amazon at a very low price.