Hi everybody
my question is about the analysis of the results. how I should analyse the data on the software and how the resultant output is interpreted? (how I should report it to others?) I have stained mice splenocytes by CFSE and divided them to three groups to set up the protocol. A) untreated CFSE labeled cells B) 3 days PHA activated cells and C) 5 days PHA activated cells. I am looking for the proliferation of lymphocytes sub-population (in this time I have not used any other markers such as anti-CD3)
I can have access to both flowjo and flowing software.
Set gates for proliferating and non-proliferating cells at 99% non-proliferating in control sample (99% CFSE positive). Then, obtain median CFSE positive and negative values. Your can normalize independent experiments according to these values.
In addition, data presentation can be done as as change in absolute cell numbers (you need to count cells or use beads while facsing), as number of divisions over time (by using median CFSE values normalized according to negative and positive controls) or as percentage of dividing cells.
I hope this helps.
You can use MFI values from treated vs control to calculate a proliferation score as shown by Ahlen et al. Blood. 2009 Apr 16;113(16):3838-44.
If you use flowjo software you can make overlapping histograms (gate your populations and then plot count vs CFSE intensity). Your proliferating cells will shift to the left of your diagram, while your control should display as a uniform peak to the right.
I second what Zengli says here. FlowJo has actually a nice in-program software to calculate proliferation indexes and adjust proliferative dyes dilution histograms.
Thank you all for your perfect guides.
Dr killie When I used the mehod suggested by your paper (I hope I have done it correctly), the proliferation score (PS) results for PHA stimulated mice splenocytes were lower than 1 e.g 0.5. Are these results expectabe? (Do it mean that my cells are divided 1.5 times?)