Resident peritoneal macrophages were separated following the traditional procedure. I used those cells to study apoptosis stimulated with LPS. Then the data was collected with FACSCalibur staining with Annexin V and 7-AAD.
I got three groups of cells showed with FSC/SSC, and I attached their corresponding Annexin V and 7-AAD profiles.
Can anyone give me some suggestions about what the three group are? (it is supposed to be two groups, one is debris, and the other is intact cells).
Hi Lei,
First I would take a step back and Answer/check the following:
1. Did you confirm by flow that the cells isolated are indeed peritoneal macrophages (MO) before you set up the assay ?If so, how pure was your isolated cells.
2. Did you check the viability of your cells (trypan Blue) prior to staining. Cell viability by trypan Blue and 7-AAD should be similar. Also did you visually look the the cells? Cell morphology will tell you a lot about what's going on the the culture.
Did you run a positive and negative control for annexin and 7-aad staining?
In your figure, are these untreated or LPS treated MO?
This information will help to figure out the problem
Hi Daniel,
thanks for your answer. To answer your questions:
1. I didnot confirm that, and i dont know the purity.
2. I did not check with trypan bule.
I do have figure untreated with LPS (attachment). Actually, If the group in the left corner is not debris cells, the result is eactly what i want.
Lei
The SSC looks a little low. Can you increase the voltage? this will give you a better look at the cell population hugging the x-axis. Your comment " If the group in the left corner is not debris cells, the result is eactly what i want"is the reason I suggested to stain with trypan blue.
I would repeat the experiment but check the sample for the items I suggested in my first response. I think that will help you isolate the problem.
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