peritoneal macrophage -protocol - (Jun/05/2007 )

Hi guys,

I'm new to macrophage and i wanted to prepare peritoneal macrophage (PM).

I have some questions that i hope you can help me with :

1. Do you use male or female mice ? and why?
2. I noticed that some people use PBS/HBSS/Medium (DMEM/RPMI/IDMD) for harvesting the PM while other use thioglycolate. what's the difference in the cell phenotype, number ant etc. ? (i wish to activate the cells so i need them as "un-active/relaxed" as possible in the beginning of the experiment.
3. in which density do you seed the cells?
4. when counting the cells, who can i know if have some erythrocytes ? how do I get rid of them ?

saying that, could you send me the protocol which works best for you ?

Thanks !

Lior.

---

For some points I can giver you an answer:

* thioglycolate is not to harverst the cells, it is injected into the peritoneum to elicit the macrophages into the peritoneal cavity, (but I don´t know if these macrphages are then activated)
* erythrocytes could be eliminated by using hypotonic lysis buffer [1.57M NH4Cl (83g), 0.1M KHCO3 (10g),1mM EDTA (370mg)]

---

1. Do you use male or female mice ? and why?
-It depends on what you want.

2. I noticed that some people use PBS/HBSS/Medium (DMEM/RPMI/IDMD) for harvesting the PM while other use thioglycolate. what's the difference in the cell phenotype, number ant etc. ? (i wish to activate the cells so i need them as "un-active/relaxed" as possible in the beginning of the experiment.

-There are 2 types of cells here.
One is monocytes, you just inject PBS/HBSS/Medium (DMEM/RPMI/IDMD) into mice peritoneal cavity to harvest non-activated monocytes.

Another is activated-monocytes (macrophages), you need inject thioglycolate or Casein to get cells activated. You must wait 3 days to get macrophages. The first day you can get neutrophils.

3. in which density do you seed the cells?
_ Dont understand your point, you meaned purification of cells?

4. when counting the cells, who can i know if have some erythrocytes ? how do I get rid of them ?
-Use Turk's solution, which stain leukocytes.
-to get rid of them, just use lysis method.

You should read search in the pubmed with the keyword: peritoneal macrophage isolation.

the method to harvest cells here:

http://icg.cpmc.columbia.edu/cattoretti/Pr...ollectPerC.html

---

hi NTH !!!

you answer was extremely helpful !!!! (the link (protocol) is great (the pictures/movie are very informative).

a couple of follow up questions :

1. gender issue - i wish to use the cell for in-vitro activation (cytokine production, phagocytosis and etc.) i fear that using female mice will render me sensitive to hormonal changes. what do you think ?

2. cell density - after you count the cells, how many cells do you seed per 1cm² (or let's say in a 6-well).

3. did you find any difference in using PBS, HBSS or RPMI ?

once again THANKS !!

Lior.

---

Hi guys,

another question... i harvested the cells (yesterday, with PBS) and look at them today. i can see 2 phenotypes.

1. cells that adhere well to the well
2. cells that are in suspension (round and bright).

Is this the usual outcome ?

Thanks,

Lior.

---

Those not adhering can be Lymphocytes.

---

how did lymphocytes got there ? there was no visible blood ?

i guess a CD11b staining will clear this issue.... what do u think ?

Lior.

---

Yes that will. Peritoneal cavity and Pleural Cavity have a subclass of B Lymphocyte. Read articles on B1 Cells.

---

Using PBS, you will get rich-monocytes, then you purify it by adhering to plastic as you did. The non-adhering cells would be neutrophils, lymphocytes or dead monocytes. You can stain these cells by Giemsa to see that.

you can harvest monocytes adhered to plates by trypsin or scrap.

---

You shouldn't have any neutrophils in the peritoneal cavity without blood contamination and there are also T-lymphocytes as well as B-lymphocytes and resident macrophages and monocytes.

Ceri

---