Hello,

Please does anyone know how I can prepare peritoneal macrophage cells for subsequent phagocytosis and hydrolysis studies?

Thank you in advance

NathF said:

####o, Please does anyone know how I can prepare peritoneal macrophage cells for subsequent phagocytosis and hydrolysis studies? Thank you in advance

You need to inject some kind of starch solution into the peritoneal vavity of the mouse, wait for 3 days. Sacrifice the mouse, inject 2-5ml of tissue culture medium into the peritoneal cavity and gently press the abdomen to bring the cells into suspension

Open the abdomen skin and hold up the center of the peritoneum with forceps.
Make a small hole in the peritoneum and remove the medium wiht the pipette.

I use 16% sucrose, sterile. Inject it and remove the cells on the same day as the previuos person described. The "starch" mentioned is thioglycolate and this is used for activating cells prior to harvest. It is not always necessary.

I've used 30% sucrose solution to obtain PECs. Carefully remove the skin leaving the peritoneal membrane intact. Inject ice cold sucrose solution (~5-6 ml per mouse) using a 26 gauge needle. The cavity balloons out, gently palpate for about 30-60 sec, aspirate out using a 20 gauge needle. Spin down, wash with medium (e.g. RPMI+5%FBS) and resuspend. count and you're ready to go with your assays with peritoneal exudate cells. Further purification can get rid of the T and B cell contaminants. Also, to enrich macrophages, you can use 2-4% thioglycollate ("age" the solution for atleast a week) injected into the peritoneal cavity, and let the mouse be for ~3-5 days - this gives "elicited" macrophages. ~10h of thioglycollate injection will enrich for neutrophils.

samm said:

I've used 30% sucrose solution to obtain PECs. Carefully remove the skin leaving the peritoneal membrane intact. Inject ice cold sucrose solution (~5-6 ml per mouse) using a 26 gauge needle. The cavity balloons out, gently palpate for about 30-60 sec, aspirate out using a 20 gauge needle. Spin down, wash with medium (e.g. RPMI+5%FBS) and resuspend. count and you're ready to go with your assays with peritoneal exudate cells. Further purification can get rid of the T and B cell contaminants. Also, to enrich macrophages, you can use 2-4% thioglycollate ("age" the solution for atleast a week) injected into the peritoneal cavity, and let the mouse be for ~3-5 days - this gives "elicited" macrophages. ~10h of thioglycollate injection will enrich for neutrophils.

Samm, thank you for your helpful comments. I was wondering if you could direct me to some literature that is pertinent to this topic. Particularly macrophage enrichment.

Al

Hello everyone,

Please does anyone know how I can prepare bone marrow derived-macrophage cells for cocultures with T cells?. but without using a cell line conditioned medium since it is not available in my lab.

Thank you in advance

kellyayon said:

Hello everyone, Please does anyone know how I can prepare bone marrow derived-macrophage cells for cocultures with T cells?. but without using a cell line conditioned medium since it is not available in my lab. Thank you in advance

I think the easiest, although not perfectly pure, is to harvest the BM and collect the monocytes by overnight adherence to plastic in medium with at least 10% serum. You'll get other cells in this prep as well, such as some mesenchymal cells, which overtime will proliferate and take over the culture. If you need greater purity, then I suggest selection with magnetic beads or sorting.

Off course anything you do to these cells, such as culture, will affect their gene expression and possibly function. Just something to keep in mind.

[/quote]

I think the easiest, although not perfectly pure, is to harvest the BM and collect the monocytes by overnight adherence to plastic in medium with at least 10% serum. You'll get other cells in this prep as well, such as some mesenchymal cells, which overtime will proliferate and take over the culture. If you need greater purity, then I suggest selection with magnetic beads or sorting.

Off course anything you do to these cells, such as culture, will affect their gene expression and possibly function. Just something to keep in mind.
[/quote]

thanks alot for your help, I will take in account.

Best regards,
kelly

[quote=kellyayon][/quote]

I think the easiest, although not perfectly pure, is to harvest the BM and collect the monocytes by overnight adherence to plastic in medium with at least 10% serum. You'll get other cells in this prep as well, such as some mesenchymal cells, which overtime will proliferate and take over the culture. If you need greater purity, then I suggest selection with magnetic beads or sorting.

Off course anything you do to these cells, such as culture, will affect their gene expression and possibly function. Just something to keep in mind.
[/quote]

thanks alot for your help, I will take in account.

Best regards,
kelly[/quote]

Just to add a short note: the above technique can be used upto about ~4-7 days for mouse cells, especially with low amounts of rmIL-2 (0.1 ng/ml, daily) added, to have enriched (but not pure) macrophages, (good enough for T cell activation assays). Try to avoid keeping them longer.

Hello everybody,

I want to do a HFD and at the end isolate elicited macropheges, using thioglycollate injection. I am wondering what are the effects of this solution on plasma parameters (TG, CHol, FFA..) and on tissues (I want to take several organes as liver, WAT...)

PLease someone, help.

Warmest wishes

Labdancer

tittletv said:

I use 16% sucrose, sterile. Inject it and remove the cells on the same day as the previuos person described. The "starch" mentioned is thioglycolate and this is used for activating cells prior to harvest. It is not always necessary.

Hi,

are the cell yield comparable to that after thioglycolate injection and do you have a reference for that ?

Thanks

I've been looking to do primary macrophage isolation for blood brain barrier studies, and found a great site that has full-length protocols: www.lipidmaps.org. If you click the "protocols" button, there are some on the harvesting and culture of primary macrophages. I haven't tried them yet (need to order and age the thio first) myself, but it's a start! One thing to note is that they do not use cold sucrose sollution to harvest the peritoneal macrophages.

Does anyone have suggestions on what kind of thioglycollate to use? Sigma's site has a bunch of broths and stuff that seem to be more for use in microbiology, and there's other stuff in it to promote growth of anaerobic bacteria (reduces O2 content). Will these additives cause problems? I also saw things like sodium thioglycollate... can these be used?

Thanks!