A protocol would be nice, but any advice would be appreciated...
The best way, how to isolate macrophages from peritoneum, is to let the adhere to tissue culture treated dish. Just make the peritoneal washing with PBS or HBSS or even medium (RPMI1640+10%FCS) and seed the cells on the Petri dish. Let the cells adhere for at least 1 hour and wash away non adherent cells. Then you have to collect macrophages with 1mM EDTA and thorough pipeting. In order to get more macrophages, inject 1ml of 3% thioglycollate medium i.p., let the mouse for 72 hours and do the lavage. The macrophages will be mainly of recruited monocytes origin, i.e. more M1 like proinflammatory phenotype. Finally isolation from bone marrow gives you highest number of M1 like macrophages.
For the peritoneal macrophage isolation, you can proceed with PBS washing the peritoneum by the 10 cc syringe, introducing it with the needle under the extremity of sternum (tip turn to down, and lift up the peritoneal shell). Incubate the sterile biological material in plastic Petri dishes at 37 °C; after 1 h, remove the non-adherent cells by 2-3 washing and incubate in a medium. The most of cells are peritoneal macrophages (the macrophages are very adhesive). By these macrophage cultures, you can study many properties of macrophages.
It needs make a specification: if you proceed with simple PBS, you obtain a resident population of macrophages, while if you use a stimulation, e.g. thioglycollate or other inflammatory stimulus, macrophages are not exactly the same.
You can find other useful details in methods reported in Cecconi O et al 1997, and others the same authors.
OK. And when we do obtain the peritoneal MFs through adherence, then how do you recommend that we proceed for the measurement of SOD? How do we do the permeabilization step (we intend to use sonification, but are unsure of the right conditions and parameters for the sonificator)? Most importantly, we need some method other then commercial kits, if you know of any...
Also, if you can point me to a paper which has described SOD measurements from MFs, that would be helpful...
My recommendation would be to be thoughtful about how you isolate your macrophages. If you are using murine peritoneal cells thioglycollate as mentioned above will give you a lot of cells but they are activated toward an M1 phenotype. Mineral oil will elicit fewer cells but they are less activated. If you use resident cells, the number is drastically reduced but they are in an unactivated state. If using peritoneal cells, they are already fairly pure, but adherence is easy and effective as mentioned above to remove non-MF cells.
I mention all this because in our experience the level of activation is going to effect the background level of SOD in your cells. If you are looking to induce SOD you would want a low background in order to measure a change.
As far as measuring SOD, we've done PCR and western blotting to look at expression levels. We've also tried a kit from Cayman Chemical that measures SOD activity. I believe it will capture all 3 types of SOD.
Good luck.
https://www.caymanchem.com/app/template/Product.vm/catalog/706002
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