Hi everyone,
We wanted to do Intracellular cytokine staining on activated T cells and need to make sure about their gate in the flow-cytometer.
We're using isolated human PBMCs and activating them with CD3/CD28/CD2- Immunocolt for 5-7 days to see the effect of some drugs on proliferation and cytokine secretion. However, we found 2 bulbs near the lymphocytes that made us concerned about them since if they were dead cells why there are 2 different cell populations?
I really appreciate it if someone could help me with this. we thought it might be related to the media and FBS that we used.
The one on the far left is formed by cell debris, the one in the middle is probably comprised of apoptotic cells (low size, increased density). So the way you've gated your lymphocytes looks okay. However, in a case like this it's highly recommended to add a live/dead stain to your protocol to be able to exclude apoptotic cells from analysis. Keep in mind that dead cells may exhibit different autofluorescence parameters and mess with your results.
Hello. If you have painted for a marker for T cells eg CD4 you can find the T lymphocytes by forming a plot with SS in the vertical axis and CD4 positive ◇ Fl "color " in the horizontal axis
Then you can identify which is the proper population in the FS/SS first image
Mehri Hajiaghayi Fatemeh Gholizadeh
Hi Mehri and Fatemeh,
Your gate is fine and you have gated on live lymphocytes.
The population that is in the middle (lower FSC and Higher SSC) harbors apoptotic/necrotic cells.
You can add a Live/Dead staining in your set up or simply perform a PI staining. You will see that the middle population will become 100% PI+.
Concerning the reason:
As you can see, the middle population has more frequency in the stimulated condition compared to the unstimulated cells, possibly showing that there might be some activation-induced cell death.
Also, there can be cell death due to lack of enough nutrients and growth factors. Do you add IL-2 in your culture medium? Do you refresh media during the 5 days of culture? What about the seeding density and plate that you are using?
Best,
Maryam
Violetta V. Vlasova , Constantina Bounia
Many thanks for your response,
We will use 7AAD to see if the middle bulb was apoptotic cells.
Dear Maryam Zare
Thank you for responding to this concern,
Our concentration of Immunocolt used for activating lymphocytes might also have been slightly high, which may explain these apoptotic cells. Our experiments did not use IL-2 as a growth factor and washing the cells during the 7 days might have been a good idea as well.
We will check the phenotype of the middle bulb in our next experiment.
Best regards,
Mehri
Hello Maryam, These extra lymphoid populations appear to be debris/cell fragments(very low FSC/SSC) and dying cells(reduced FSC and greater SSC). You can confirm this easily by the addition of PI and looking at a bivariate plot FSC vs PE(PE-TxR, Pe-Cy5, Pe-Cy7) fluorescence. Population just below lymphocytes should light up with red +++ fluorescence, the very low FSC/SSC population is too damaged to bind DNA dyes so will be mostly negative +/- red . Hope this helps.
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