I am going to observe the dynamic change of TAM in B16 melanoma, my current gating strategy is CD11bintF4/80hi, but the cell cluster is not very clearly. Is this strategy accurate enough? How to distinguish TAM from monocyte and tissue resident macrophage?
If your separation in flow is poor you can change the fluorophore to something brighter, increase the laser voltage on that channel, ensure you're not overcompensating, and removing colors causing spectral overlap.
You can somewhat remove monocytes in your forward x side scatter gates but gating on CD14- events will be better. Some would argue that tissue residents macrophages found in a tumor are TAMs by default, but if you mean to identify M2-like suppressive TAMs then CD206 expression is a good correlative marker and Arginase I is a good functional marker.
Good luck
Dear Xiang An
I just briefly wanted to expand on Brett P Gross points. His flow cytometry advice is extremely sound and makes a lot of sense!
In addition, you may wanna, check out this publication by Micke Karlsson's group at Karolinska Institutet.
Article Reprogramming Tumor-Associated Macrophages by Antibody Targe...
He uses the B16 melanoma model to characterize tumor associated macrophages (TAMs). They stain live CD45+ CD11b+ cells and define inflammatory monocytes, immature macrophages and M1 and M2 TAMs based on Ly6C and MHCII expression. Please see figure 1D for flow cytometry graphs and gating strategy.
I hope that helps!
All the best & good luck with your experiment,
Michael
Dear An
Look at
Article Tissue-Resident Macrophages in Pancreatic Ductal Adenocarcin...
I think help you.
Good luck
Hi Xiang
Try to play around the voltage. sometimes the cells are there and everything is fine regarding the procedure but you can not see the population that you want.
Best wishes
Shilan
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