Hi
I would like to measure apoptosis in murine neutrophils isolated from blood on flow Cytometry
i am using the Annexin/PI kit from Invitrogen
Initially, I would like to see what is the percentage of isolated cells are apoptotic ( Annexin V positive) and necroptotic ( Annexin V and PI positive)
I don't think I need to stimulate the cells to induce apoptosis, but I definitely need something for the positive controls.
Does anybody know something I can use to stimulate the cells?
Something short term.
Also, what would be a good choice for compensation controls for this experiment.
If anybody can recommend any publications on the subject, that would be highly appreciated
There are two protocols. One ir with H2O2 and the other with UV light. This paper is excelen for you.
You can please refer to the link below to obtain certain valuable informations:-
http://www.jleukbio.org/content/72/4/685.full
You can also download pdf of the file attached herewith.
Easy, following short PMA (200-500 ng/mL) stimulation, they get hyperactivated rapidly and then commit to apoptosis. We tried that out in primary human neutrophils.
However, this type of apoptosis has a special name: NETosis.
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3193439/
http://www.jleukbio.org/content/70/1/52.long
***
It seems natural for neutrophils to go into apoptosis after going into an activated phase and getting their job done. See a review
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2956014/
***
For a blocker control, since PMA is a well known PKCs activator, you can include a PKC inhibitor to block off the effects.
http://www.tocris.com/pharmacologicalBrowser.php?ItemId=164170#.VfxMId-qpBc
The "Go" series of PKC inhibitors (see above link are usually very effective at 50 to 100 nM.
- Badges
- Science method