Hi,
I'm trying to asses the electroporation efficiency of monocyte derived dendritic cells that I differentiated from PBMC monocytes.
As you can see from the picture attached, after FACS 4 distinct populations form in SSC x FSC. I'm not sure which population I should gate for the monocyte derived DCs in order to exclude other cells & debris. I tend to use the one with the circle around, however it's only 20-30% of all cells which seems a bit too little.. Would you include population A maybe as well?
B & C are according to the dead cell marker pretty dead.. A is half half. However, nearly all successfully transfected cells are in the circle population.
Thanks for your help!
Hello Sven Be,
According to your attached density plot, i can only look at the FSC vs SSC cells. You haven'r shared death cell marker pics. Still, i think, you need to take population A into consideration.
Best regards,
Babu
I would use antibodies against surface markers such as CD11c and HLA2. This will discriminate the DCs from non differentiated monocytes, lymphocytes and dead cells. But with just this data your population should be in the circle, you could even expand it a little, do FSc vs dead cell marker (instead of SSc) and it will become more clear. At an educated guess: C are dead cells, B are lymphocytes, A are undifferentiated monocytes (maybe some B cell doublets). But I would highly recommend using the antibodies in future
1. Firstly, search the literature to find the suitible dendritic cell markers for your research. You can choose the following moAbs such as CD11c, HLADR, CD80, CD83, CD86, CD85d, CD123, CD1c, CLEC9A, etc.
2. After the choosing suitible moAbs. stain your sample.
3. Search the positive population in the logaritmic Florescent Plot and then gate it. So, you can find your right population on the FS vs SS plot. (Back gating)
4. You can also stain with monocyte markers such as CD14. You can choose the CD14 negative population on the CD14 vs SS/or FS plot. So, you can eliminate the undifferentiated monocytes.
I think like Dr. LC Davies, C is debris, B is Lymphocytes and A is undifferentiated monocytes. Your target cells may be in the circle. I think they are more bigger cells than lymphocytes and monocytes.
I think as Dr. Mehmet Arasli. Normally MMDCs are bigger cells than mono. How long did you cultivate then? 5-7 days?
According to my experience, after electroporation, monocyte-derived DCs tend to decrease in size, and might be as small as monocytes. As already said, it is essential to use antibodies (CD14 and CD11c might be a good combination) to determine your true population.
20-30% of live DCs after electroporation is indeed low, but this cell type suffers a lot when electroporated. It might be possible to increase this percentage by trying to change the parameters of the electroporation.
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