splenocytes are not good for CFSE test because they are not easy for activation, you may try those from lymph node

Hi Lilian,

By the looks of your data, I don't see you having any problems. Your cells are responding to both stimulants. Don't be worried about not having beautifully separated peaks for each sub/daughter-generations. People seldom see such nice resolution. However, after having a closer look at your data, I have few recommendations

1. Define the highest CFSE intensity (ie, unstimulated cells) to the right most of your X-axis. This can be done by adjusting the PMT/voltage of your FL1 channel. this will provide a bit more room to work with the sub populations.

2. Use a third party software for analysis. In my experience ModFit does a wonderful job. Just ensure that, you define the position of the parent generation properly for each of your samples.

Good luck!

Jose

For better resolution of peaks, try the following:  First, an extra wash in PBS prior to CFSE loading can help to wash away any residual protein (BSA, serum) and give you more even / consistent CFSE loading.  Second, a lower dilution or shorter time for CFSE loading of your cells.  A final note is to collect just 5,000 - 10,000 events on the flow cytometer ... a higher number of events can make your CFSE peaks look "blurry."

Hi Lillian,

I frequently use CFSE dilution as a read out of Treg suppression, and if I may make a few suggestions;

1) I agree with Jose, that adjusting your PMT voltage (for the FITC channel) so that your positive control sample (unstim'ed) is on the far right on the X axis, will help you as you have quite a few events stuck on the y axis because they are so low on the FITC scale.

2) 2 e 6 cells/ml is quite a dense population of cells to expect robust proliferation over 5 days.  I would recommend titrating that down so that your cells have room, so to speak, to expand in culture.  

3) T cells prefer cell-to-cell contact, and I find that V-bottom tissue culture plates work more effectively than flat bottom to promote this interaction.

4) I cannot ascertain the degree of apoptosis per condition based in the assay file you provided.  If possible, try adding a live/dead dye like Zombie-Texas Red so that you can discriminate your live cells in your analysis.  

5) As for "blurriness", sometimes that arises from non-uniform staining of your sample. I wash my samples in PBS, resuspend in 500 ul PBS, add an equal volume of PBS containing 2X CFSE, but add it while vortexing the sample on medium speed for 15 seconds.  After that 15 seconds is done, I incubate the sample at RT for 5 min, and quench with pre-warmed media in excess.

Hope these suggestions help you and best of luck!! 

Thank you all for your advice, I believe I have finally figured it out. Here is what I did:

During, CFSE staining I reduced my working volume to ensure a rapid and uniform staining and set my PMT voltage to the far right and collected only 10 000 events.

Because, splenocytes are a heterogenous population I decided to separate out CD3 positive from negative populations . CD3 positive cells proliferate better than other cells types i.e. monocytes are believed not to proliferate. This allowed me to observe nicer peaks. See attachment !

Furthermore, I had additional questions:

Shannon, at what density to you seed your cells in v-bottom (96WP?) and for how long do you carry out your proliferation assay?

Hi Lillian,

Your CFSE profiles look great!  Glad you could get it to work!

I apologize for not responding sooner - I only just saw your question today!  I allow 72 hours of proliferation for my assays, and I plate 20,000 responder cells per well in a 96 WP format.  That should calculate out to 1 e 5 cells/ml.  That said, since you're doing 5 days in culture, I'd use a number around that range to accommodate the growth.

Let me know what you try!  I'll try to be better about checking responses!

Shannon