LPS (1ug/mL)-stimulated microglia are positive for CD68, but so are Vehicle (serum-free medium) treated microglia. I used fluorescent microscopy to visualise them. Fluorescence intensity for CD68 in both groups show no significant difference! So I'm skeptical about using anti-CD68 antibodies to identify activated microglia. Please suggest where I could have gone wrong.
Hi, Where are your antibodies from, are they being used for FACS? CD68 is a marker for macrophages, DCs and microglia. Microglia express this in both the activated and incactivated state - how long are you maintaining your cells before analysis on FACS? - Could there be a tendancy to auto-activate over time?
Also, do you see a shift in the histograms that is not coming through in analysis, or is the visual shift also unclear? I wonder whether it could be a case of analysis rather than results? Best wishes and good luck, Brittany
Hi,
As mentioned by Brittany CD68 is generally considered as a phenotypic marker and its use to study activation is bit skeptical. You may want to consider using other markers such as MHCII,CD40 or CD86.
Thanks Brittany and Shinsmon! The antibodies are from Abcam. I performed immunocytochemistry and captured images using confocal microscope, no FACS involved! Microglial culture was maintained in a serum-free medium for three days prior to LPS-treatment. Immnostaining for CD68 was performed 3 days after the treatment. TNF-alpha ELISA indicates the release of the cytokine from LPS-treated samples, which is an indicator of activation. However, the morphology of microglia and CD68 expression do not correlate with the activated state of microglia. So, I am more tempted to trust ELISA data for activation state rather than morpholgy or CD68!!! Can you suggest me a paper that talks about CD68 staining as I need to cite one for my report!
Many thanks!
Hari
Activation of microglia is a delicate issue. More than a discrete state, the activation of microglia is rather represented by a continuum of states. In my opinion, it largely depends on the authors how they particularly define the "activation". Nevertheless, to my experience, microglia undergo even the morphological changes according to stimulation; also see attached link.
http://www.ncbi.nlm.nih.gov/pubmed/1907214
There is no single test to identify "microglial activation", because there is not uniform state called activation. Microglia have different activation phenotypes, which are linked partially conflicting functional features (Dr. Carol Colton's has couple of enlightening reviews about this). Different stimuli induce different responses and surface protein expression might not always reflect on those. E.g. LPS typically will induce NO release, some pro-inflammatory cytokines and very distinguish morphological transformation. I am not sure what happened to anti-inflammatory cytokines or trophic factor release with LPS, as I rarely use it myself. In your situation, LPS did not change CD68 expression, but it might be that CD68 basal expression of your primary(?) microglial cultures was elevated due to serum-deprivation, or simply it just is not sensitive method to detect "microglial activation". My suggestion is that you identify several functional outcomes typical for your stimuli (i.e. LPS) in your culture conditions (i.e. serum deprivation) and then see how those are changed with what ever treatments you plan.
One more point, in papers people (including myself) use the term "microglial activation" in lack of the better term, but nowadays referees usually expect multiple outcome measures, particularly in vitro, in vivo is still a pickle...
I call microglia the drama queen of the brain, so on that note - Good luck! ;)
Hi,
recently, an article was published showing that the phenotypic change of microglial cells by modulation of the mtor system causes an expressive increase of CD68 without the expression of proinflammatory markers, although this state cause epilepsy, astrocytosis and microglial proliferation. Taking into account that activated microglia does not necessarily mean that they are expressing proinflammatory factors, and these cells react in a very particular way depending on the nature of the stimulus that is applied or experienced, my suggestion is that you carefully analyze the nature of your experiments to not fall into methodological pitfalls, especially those related to choosing appropriate markers, specially because define activated state is far to be a trivial task.
Good luck!
Hi Hari Sapkota ,
I have the same experience as you. I saw some papers show that LPS-stimulated microglia/macrophages increase their CD68 expression dramatically. However, I tried two difference CD68 antibodies, but both unstimulated cells and LPS/IFNg stimulated cells (6h and 24h) show similar level of CD68 expression by fluorescent staining. FYI
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