I am trying to use monosodium urate for immunofluorescence as a positive control in macrophages, however since they are insoluble crystals, I am trying to wash off the crystals not internalized but many still remain bound to the dish or non specifically on cells.
Any suggestions to overcome this issue would be helpful.
Removal of unbound crystals from the surface of macrophages is quite straightforward with three washes with ice cold PBS supplemented with 5mM EDTA (remember that EDTA needs to be made up as a 0.5M stock solution first with warming and pH adjustment to ph 8.0 so it goes into solution). The washing protocol is described in our paper describing macrophage phagocytosis of MSU crystals in 2000 (Yagnik DR, Hillyer P, Marshall D, Smythe CD, Krausz T, Haskard DO, Landis RC. Arthritis Rheum. 2000 Aug;43(8):1779-89). Good luck with your experiments.
If you just want to use the macrophages for staining a single 15 second (!) wash with ice cold 0,1% formic acid in water will work. after 15s of incubation add 20 volumes of PBS to neutrolice the aciud
Another way is to use tris buffers or sucrose, which break up H-bonding:
see:
Freedman ML, Weissmann G, Gorman BD, Cunningham-Rundles W.Sickle hemoglobin gelation--inhibition by tris (hydroxymethyl) aminomethane and sugars.Biochem Pharmacol. 1973 Mar 15;22(6):667-74.
Also: make sure you have crystals (via X-ray diffraction), not amorphous urate.
Good luck!
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