to me this sounds like hypersignaling of AKT which is known to induce apoptosis. But why would you need to induce apoptosis? It may be more relevant to check just baseline apoptosis levels. You may also want to look at upstream (PI3K) molecules of AKT and also other related molecules such as PTEN or also ERK. It would help you to understand if this effect is due to AKT alone or a general signaling phenotype. a 4G10 blot could be very useful in that regards.

Should your hypothesis be that deletion of Gene X causes increased signaling through AKT in physiological conditions of cell population A then indeed the methods you suggest are a possibility. Just be aware that sorting cells for instance to get a specific bone marrow / HSC cell type will cause itself cell stress and thereby increased apoptosis. cleav. casp3 can for instance also be measured by Flow to my knowledge although I never tried it myself. There are numerous of stainings, for instance you could try IHC (which could allow you to stain bone marrow populations) together with apoptosis markers (tunel staining, cleav casp3).

Best

BK

Dear Christian,

I agree with Behzad to study the natural pathway of your KO model with increased AKT signalling, that could give some responses for this pathway without conditions of stimulation.

Even if I am far away from fundamental intracellular signalling, I have indirectly studied this pathway (see our paper on my wall on GAS6 genes and myocardial infarction). Upstream of AXL/AKT pathway, one naturally way of activation is TAM receptors (TYRO3-AKT-MERTK recpetors) which are effector of GAS6 protein ; for the product of Growth Arrest Specific Genes, first studied in cell proliferation, then for their implications in ahterogenesis during stoke and acute coronary syndrom. And in these pathologies, the supposed mechanism is effects of GAS6 protein on vascular cell proliferation and increase apoptosis of cells in the atherosclerosic plaque, that can lead to necrosis and rupture.

So, a model of permanent activation of AKT pathway (if I have well undestood yours) can be put in parallel with some natural mechanisms, but also some human pathologies.

Best regards

Didier

It is true that sorting itself would create stress. But if you want to be sure that you are dealing hematopoietic cells, I think you have to sort. Make sure to stabilize them after sorting and then you can give induction to apoptosis (if Akt involvement has to be checked, then your method may be correct) and then checking for apoptosis markers in the whole cell population should do.

Dear Christian,

if you only need a method to check and quantify apoptosis, i will send you a paper where the amount of apoptosis in pre-nervous NT2 cells was checked by cell fluorimeter:

Apoptosis as a specific biomarker of diazinon toxicity in NTera2-D1 cells.

Aluigi MG, Guida C, Falugi C.- Chem Biol Interact. 2010 Sep 6;187(1-3):299-303.

I studied apoptosis of human haemtopoietic stem cells following cryopreservation by using Annexin and 7-AAD, it works very well and gives interesting information.

We have isolated and expanded HSC from mice for cell death analysis - see e.g. Labi et al G&D 2010. A problem with this is that druring expansion to get enough numbers for FACS, HSC may loose HSC characteristics. So always co-stain for lin markers when using AnnexinV.

The most powerfull assay to test impact of loss of function of your gene of interest is to do mixed bone marrow chimearas using the Ly 5.1/5.2 system (see eg. Labi et al 2013, EMBO Mol Med) and monitor reconstitution over time in the blood. You can also do serial transplants after 16 weeks if you do not see any impact

If this is not an option, isolate LSK cells by FACS or Fetal liver and perform Methocult assays (Stem Cell technologies) in limiting cytokine concentrations or upon your favorite treatment.

Hope that helps.