I need to analyze the TNF-a production from mouse macrophages from brain by Flow cytometry. I see there are different protocols used by people. All I see is the addition of Brefeldin A to the cells prior to staining. I want to know how and at which step I need to add the Brefeldin A to the cells prior to staining. Do I need to stimulate the cells with LPS or any other ligand like we do PMA/Ionomycin stimulation for T cells?
Dear Tabasum,
Please check the attached paper for a protocol that lays out intracellular cytokine staining in brain leukocytes. As there is only a minimal amount of leukocytes infiltrating in the brain (in that particular model of neuroinflammation), it was rather difficult to establish intracellular cytokine staining. Therefore, I'd advise to re-stimulation and LPS, but maybe depending on your model, something else might be more suitable.
Concerning your Brefeldin A question, I had great results with Golgi Stop/ and Golgi Plug (containing monensin/ Brefeldin A), however I would advise that you restimulate your macrophages AND add Brefeldin A (for a minimal amount of time) as it strongly influences the expression of surface antigens (like F4/80 or others), you wanna use for macrophage identification.
Article A role for the histone H2A deubiquitinase MYSM1 in maintenan...
All the best and good luck with your experiments,
Michael
Hi Michael,
Thanks for the info. I really appreciate! Mine is the strong brain inflammatory model. It is the TMEV (virus induced brain inflammation) which leads to the prominent infiltration of cells into brain. My concern is do I need to restimulate the cells or just proceed with Brefeldin A treatment of cells.
Hi Tabasum,
I'd suggest (and assuming) you compare two mouse strains (resistant vs. susceptible) with a qualitative difference in survival, I would restimulate with LPS or a Tlr7 or Tlr9 ligand for a short period of time (one to four hours) adding Brefeldin A, one hour before you take the cells for your FACS staining and then just follow standard staining procedure of live dead stain, extra and then intracellular staining. You might have to optimize the duration of the restimulation with a time course, but it's basically the standardized setup from my experiment. I hope that helps!
All the best & good luck,
Michael
Hi Tabasum
For intra cellular staining the use of monensin/ Brefeldin A is the better method. I´m agree with Michael, maybe you need diferent condition with or wtihout stimulate of Ligand for TLR´s. The use of PMA/IONO it´s more for T cell stimulation
Good Luck!!
Hi Tabasum ,
as you mentioned that you´re using a rather strong brain inflammatory model, you may consider to avoid TLR restimulation (that harbours the risk of generating false positive signals), but trying to apply the Golgi blockade in vivo. There are several papers describing systemic Brefeldin A injection into mice (PMID 20660615, 17371998 , 17372004 , 15879085 etc)
I used 250µg Brefeldin A per mouse injected i.v. 6hours prior to sacrifice (worked pretty well for Il-6 ex vivo FACS staining in peritoneal macrophages
Good luck
The real issue is to discriminate macrophages from microglia cells. Also you need to do all the steps at 4C to avoid not specific activation and you should also perfuse your animal. Please have a look at this paper: Article Local Cues Establish and Maintain Region-Specific Phenotypes...
In this paper you can also find antibodies that work well for microglia. Please be careful with the fluorochrome that you choose. Some of them are not giving you good results (such as fluorochromes from the red laser) in old mice because there is an increased background fluorescence.
- Badges
- Science method