If you see that effect for all your unstimulated cells, it can be something from in media etc. But if you just see the proliferation in some replicates (cells isolated from one individual), it is probably caused by the biological variations. I saw in my experiments that some cells from one individual do not respond to stimulant, (e.g. they have the high level of cytokines without any stimulation ). It can happen as a result of some internal pathogens. You can isolate the cells from individuals separately and check if you have any biological variations. 

Hi Kate, 

Here are my suggestions:

1- It could be that your staining protocol needs to be tweaked. If your cells are not staining well with the CFSE then you might be getting a weak signal. A good control would be to fix cells (4% PFA) right after staining and run it on FACS to detect signal. 

2- As khalil mentioned, it could also be a contaminant in your media that is causing polyclonal expansion of immune cells.  but doing what i suggested in the step above will help answer that as well. 

3- It could also be that your stimulation is not potent enough (hence no difference between stimulated and unstimulated). Do you have access to anti-CD3/CD28 beads?   

Let me know if you have any further questions and i can help you trouble shoot. 

Hi Kate, 

What do you mean specifically with your statement, "CFSE signal from my unstimulated cells is around where my stimulated cells are?" Do you see a single fluorescent peak, or do you see multiple fluorescent peaks in both stimulated and unstimulated? If you see only a single peak, it definitely means the cells have not divided, and thus have not been stimulated properly, or are not being cultured properly. If you see multiple peaks, that indicates division, and then you have a problem with the unstimulated cells being induced to divide. These are the basics of CFSE dilution assays, so you probably know them already, but I just want to make sure we aren't missing any technical details.

The concentration of ConA that you are using seems extremely high. I have tried a range of concentrations on human PBMCs, 2 ug/mL is the lowest to stimulate division, 5-10 ug/mL is a strong division signal, and 20 ug/mL is actually toxic - it kills most of the T cells and they do not divide. If you are adding 1 ug / 20 uL, that is 50 ug/mL, which I would certainly predict to be toxic. I cannot absolutely say that it would be toxic to mouse T cells, because I haven't tried that high of a dose with them, but I do know that around 2-5 ug/mL is sufficient to proliferate mouse T cells as well. 

If proliferation of the unstimulated controls is the real problem, then Simon and Khalil's answers will be really helpful. I have never experienced that RBC lysis activates mouse T cells (we use standard ACK buffer). Try using a different batch of FBS. If that doesn't work, try resting your mouse PBMCs in media overnight before you label them with CFSE. And if that doesn't work, get some PBMCs from another investigators mice because you might have an infection in your mouse colony, and I sincerely hope with all my heart that is not the case!!

Good luck and feel free to respond with any more questions.

Thank you Simon,  I feel like it might be my CFSE staining but I have remade my media just in case.  I believe I do have access to beads but I have never used them before.

I want to do a titration with my CFSE to see what works best, some concentration between 1 and 5 uM seems to be most recommended.

Excuse my ignorance but what is 4% PFA?  Since I've only worked with surface markers I've never had to fix any of my cells before

Dear Kate,

I would not use RBC lysis to isolate PBMC. Heme is known to activate monocytes and to induce inflammation, though the inhibition of this molecule is what is correlated with enhanced proliferation of T cells.

I have one concern: are your reagents LPS-free? If not, you should use Polymyxin B in order to verify the possible influence of LPS contamination in your protocol.

Another important question raised by Khalil is whether you know if your blood donors are healthy. Sometimes, a person looks healthy but is recovering from an infection (maybe a flu) and its immune cells are still activated and proliferating. If you tested this protocol only once, I´d recommend you to try again, this time using additional points with Polymyxin B . 

Kind regards,

Raphael

Hello Kate,

I have used in the past 1μΜ CFSE to stain mouse peripheral T cells and used 5μg/ml ConA for 72h. CD4 cells perform multiple divisions within that setup, whereas CD8 perform one synchronized division. I believe you over-stimulate your PBMCS for a prolonged time, which can actually lead to extensive cell death. Have you checked any activation markers eg CD69 and CD25 for T cells within the first 24h of the stimulation to ensure your cells are activated in the first place?I would also monitor CD4 /CD8 divisions for T cells separately gating on TOPRO-3 negative compartment. You can also perform staining in the CFSE labelled cells for B cells etc as well.

Furthermore, I wanted to ask you how you perform your CFSE staining protocol. When it comes to culturing conditions, I normally used RPMI‐1640 supplemented with 10% FCS , 1% P/S and L-glutamine

I hope I helped a little!

Good luck!

PFA is paraformaldehyde.  Let me know how it goes.  

So I just reordered the CFSE - I was using aliquots another lad made had made and I'm unsure of how she split it up, there is a chance the concentration was off because of that.  Right now I just stained some cells with Cell Trace Violet and I'll see how that works.

Also, I might have made a typo but I am using 5ug/mL of ConA (1ug in 200 uL) so not 50ug, that would be alot.

I will let everyone know how the new stuff works.  Thank you for the suggestions

Hi Kate, working with CFSE was a bit tricky for me initially, but here is what I learnt and I hope it will make your troubleshooting process much shorter.

• Label cells with 10uM CFSE (make sure to follow the manufacturer’s instructions regarding cell density, incubation time and temperature, solution recipe, washing steps-These are very critical for successful labeling)

• Tip: You don’t want the cell concentration to be too high because the labeling will not be homogeneous, also you want to quench/wash well after staining to make sure free CFSE is washed away (high CFSE can be toxic for you cells).

• Once you stained and washed your cells, allocate 250,000 cells /well in a round bottom 96well plate for cell culture. At this point, make sure to keep some CFSE labeled cells in a separate tube and store it at 4degC for as long as you are stimulating your cells (Protect the tube from light). This will be your control sample for CFSE (see below).

• On the 96 well plate add 1ug/ml of ConA –mix well and incubate at 37degC for 3 days. Do not add ConA in the unstimulated controls.

• After 3 days, collect cultured cells , wash them and resuspend them in you flow cytometry staining buffer. Do the same for your samples that you stored at 4degC.

• Stain all samples with a viability dye to assess their viability. I use the fixable viability dyes from life technologies.

• When you run your samples for flow cytometry, make sure to collect as many live cell events as possible (5, 000-10,000 events is a good number).

• For your analysis, first gate on life cells and then open the CFSE gate to see the peak (s). Your fridge-stored CFSE labeled cells will give you one peak in the far right side. ConA stimulated samples will give you multiple peaks (in total 7-8) with decreasing FITC intensity. Your unstimulated samples might give you one or two peaks closer to the peak that your fridge-stored cells will give you. Because you are using viability dye you will be able to get nicer and sharper peaks that will help your subsequent analysis a lot.

This protocol works beautifully for me.

Good luck,

Antigona

Thank you everyone for the responses but I'm still having a lot of trouble and I'm very confused.  I just did a titration of the dye, thinking that maybe I wasn't using the optimal concentration and I used 05, 1, 5, and 10uM dye.  I fixed some of the samples in 4% formaldehyde the day of staining and found that when I ran them on flow the MFI of the day 0 CFSE staining was really low and there wasn't much of a difference between all the concentrations like I imagine there should be.  I'm racking my brain trying to figure out why this staining protocol won't work for me.  I recognize that it is difficult to get very nice separate peaks from a heterogenous population but I'm not seeing a difference at all and now I'm concerned that it's because my initial staining is too low but at 10uM concentration that sees really unlikely.  I'm just a bit baffled on what to do next.

Ok, so I have a very dumb question now too.  I am using invitrogen's CFSE, it comes in 10 tubes that have CFSE that are then re-suspended in 18uL of DMSO each to make 5mM concentrations.  If I take out only 1uL of the CFSE stock is it still good to use after I re-freeze it?  That's what I've been doing but I just read an article saying that once it's thawed the rest of the stock has to be thrown out because it will hydrolyze and won't be good anymore.

Hi Kate, I wouldn't fix the cells after staining them with CFSE (see the protocol that I added a couple of days ago). Also, I wouldn't freeze and thaw CFSE for more than two times (I use the same reagent as well and never reuse it after the second time of thawing it).  Hope that helps :)

Hi all,

I am facing the problem in CFSE labeling too. I want to check the proliferation of purified CD8 T cells. For that I labeled these cells with three concentrations of CFSE (3, 5 and 10 uM) and co-cultured them with irradiated APCs on anti-CD3 coated plate (24 well plate) for 72 hours without adding any mitogen like con A. After running my samples on flow cytometer, I am getting two peaks for all the samples. In some samples I am getting one large peak on the extreme right and one small peak on the left side. However, for some samples (with relatively less cell number), I am getting large peak on the extreme left and very small peak on right. I am confused with the pattern of these peaks. Can anybody point out what can be the problem? I would appreciate.

Hi Tabasum, 

I have a few questions before i can try to trouble shoot. What is the purpose of using multitple CFSE doses? I have always used 5uM and it works well. Secondly, what is the point of adding the irradiatedi PC? Are they loaded with antigen? . IT is likely that the peaks on the left side are APCs but this depends on what your gating strategy is on FACS. Thirdly, how many washes do you do with the after coating plates with anti-CD3? it is extremely important that you was away all unbound CD3 because they may inhibit rather than activate the T cells if they are not plate bound. I also think you should IL-2 or anti-CD28 to your culture, this always helps. I am more than happy to take a look at your detailed protocol or CFSE dilution FACS plots to give you a more detailed and accurate response. Just in box me or email me [email protected]

Simon. 

Thanks Simon,

I have e-mailed you the detailed protocol along with the FACS plots. Do let me know if you have any other query regarding the protocol or FACS plots.

Tabasum

They are freshly isolated mononuclear cells (predominately lymphocytes)