Hi everyone, I'm a newby here. I need to pick on your experience.
I'm working on Monocytes and in all my experiment I found a result, that I can't explain.
After the separation the monocytes look like this (FACS - Image 1); then I put them in culture for 72h (@37°C, in Medium VLE RPMI 1640 with 2g/l NaHCO3 with 10% heat inactivated FBS and L-Glutamin (2mM)). After the culture, the FACS result are the one showed in Image 2. There is a loss of volume and a loss of CD14, almost like they are dying.
However, if the monocytes (of the same separation batch) are stimulated with cytokines (or with LPS), they look like this (FACS - Image 3).
Have someone experienced something like this? Or has someone some possible explanation?
I'm looking into my protocol to see if I do something wrong, but at the moment I didn't find much...
Thank you very much in advance!
Hi there,
What isolation method for monocytes are you using?
I am also a beginner, but as far as i know, monocytes don't survive very well without stimulation in cultures. Some monocytes die without stimulation and some will differentiate into dendritic cells (1). The downregulation of CD14 has been described for cells transitioning from monocytes to immature dendritic cells, so there may be the answer to your question.
As for why LPS-stimulated monocytes don't suffer the same CD14 downregulation, it is because LPS is recognized by CD14 molecules and LPS can actually even upregulate CD14 expression. Also LPS has an anti-apoptotic effect which may explain the higher survivability seen in your LPS-stimulated samples (2).
(1 - Material and methods section)
https://www.atcc.org/~/media/Attachments/Application%20Notes/In%20vitro%20differentiation%20of%20macrophages%20and%20dendritic%20cells.ashx
(2 - First paragraph of the introduction and the Discussions section may be relevant to you)
https://www.nature.com/articles/pr2001162.pdf?origin=ppub
Also, an easy and practical way for checking your cells after 3 days in culture is microscopy. Maybe looking at your cells through a microscope could help troubleshooting your problem?!
Hi
I think what you are seeing is probably not a loss in CD14 expression but rather monocytes dying in cell culture after 72h. Your stimulation probably boosts their survival to some extend. I didn't see a viability staining in your plots. Did you include a viability dye to your flow cytometry panel to check if cells are alive?
Yes, in the absence of growth factors, monocytes will start to lose their markers, including CD14, CD4, and CD64. This is part of their differentiation towards fibrocytes.
@ion thanks for the answer. I'm using MACS columns. Yeah, the cell culture seemed a little "stressed" under the microscope...
@oliver I didn't think about adding a viability marker. I'm adding Annexin V for the next experiment. What do you think??
Thanks @sergey that's what I thought...
@serena, you could add Annexin V if you are interested in apoptitic cells. In general, to analyse cells after in vitro cultures where you can expect some level of cell death its always a good idea to add a real viability dye (such as PI, 7-AAD, or a Fixable viability dye sold by different companies). For analysis you can exclude these cells as there is increased unspecific antibody binding on dead cells. Good luck with your experiments!
Yes, Monocytes starts to loose their surface antigen (CD14) after 2-7 days while in culture. Maintianing monocytes in culture is difficult as they start differentiating into Macrophage or DC or fibroblasts after 2-3 days in culture.
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