Dear colleagues!
I’m working with mouse splenocytes now to elaborate Treg suppression assay. But I have a trouble at the CFSE staining step. After my initial staining of total splenocytes or some sorted populations of cells from them (naïve CD4 T-cells), next day I observe the initial peaks of CFSE shift to the left with the loss if CFSE intensity ten times. So, it is not a first division when the CSFE intensity should drop 2 times only. It happens in a sample stimulated with CD3/CD28 beads and in a non-stimulated sample. Further 3 days afterwards I observe distinct division CFSE peaks in a stimulated sample. But I do not like that initial CFSE shift.
First, I thought that it was due to unsynchronized cell cycle of splenocytes and tried to synchronize them before CFSE staining keeping the cells not in a full media (RPMI + 10%FBS + 50uM ME + 25mM HEPES + 1mM Sodium Piruvate + 1% Glutamax + 1%Pen-Str) but in a media deprived of IL-2 or in a media with low 1%FBS or in a media with 1%FBS and without IL-2. Nothing changed. Next day my initial CFSE peaks again 10 times shifted.
Where is a mistake? Or is it a standard phenomenon? I add a file with the demonstration of that shift.
My protocol of CFSE staining is as follows: I wash cells with (DPBS+0,1% FBS) two times. I know that someone recommended to get rid of any traces of FBS at this step but others recommend to add FBS to splenocytes as they are very sensitive cells and quickly die without any FBS. Then I stain them for 5 minutes at 37C again in (DPBS+0,1% FBS) and then wash with cold RPMI with 10%FBS – two times.
I appreciate it greatly any tip and possible explanation!
Dear Marina Yu Loguinova
I hope you are doing well. I performed a CFSE assays using peripheral blood leucocytes. It was difficult to analyze the cells after few days of culture. I finally got proliferation data only after seven days of cell culture. Leucocytes do not have long in vitro culture lifetime.
You said that your non-stimulated cells are presenting CFSE peaks, this is unusual to happen. A non-stimulated group will present a CFSE peak similar as day 0 or small peaks of 1 or 2 divisions, while the stimulated group will be similar as you found in day 3.
I am not sure if unsynchronized cell cycle is the problem. I guess the splenocytes are differentiating in T cells during the cell culture. As CFSE is a cytoplasmic staining, the reduction of the cell size could interfere in the reduction of CFSE signal in your cells.
Looking the pdf file, you have one stimulated and non-stimulated for each day. I am not sure if you need that much for each group. Try to standardize your assay using only one non-stimulated group with the replicas and analyze all your samples at the same day or in two time points during the cell culture. Also include a non-stained cell control.
I believe you are using FlowJo to analyze your data, and in the proliferation tool of the FlowJo, the non-stimulated group stained with CFSE is usually used to mark the peak 0.
Regarding to FBS in the PBS, look at the data sheet of the CFSE manufacturer. The time of staining is short for me, I remember to stain during 15 minutes in the dark in PBS Buffer.
Sorry I am not able to help more.
Kind regards. ^^
Dear Tulio Teruo Yoshinaga,
Thank you very much for your detailed comments. I absolutely agree with all of them and adhere to them.
Now I came to a temporary solution to stain responder cells (CD4+CD25-CD127+) with CFSE and leave them in a media with only 1% FBS and without IL-2 for night to synchronize cell cycle as it was done in paper https://pubmed.ncbi.nlm.nih.gov/26975663/ Next day I’ll stimulate cells (with leaving some of them not-stimulated for control).
Dear colleagues,
The matter is explained in the paper attached. CFSE peak shifts due to the natural efllux of CFSE-bound proteins from the cells or degradation of some short-lived proteins. This substanial (more than 10 times) CFSE intensity loss is proliferation-independent. Thus, it is recomended to stabilize CFSE-stained cells in culture for approximately 24 hrs prior to starting any tests with the cells. Pay attention to Fig2 and Notes 20-21
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