Macrophage isolation and stimulation questions. - (Jul/27/2008 )

Hi, I have just started work that requires me to work with macrophages and I have a few questions to ask.

I have been hearing things about using an ice-cold 16% sucrose solution to isolate primary macrophages from mouse peritoneal cavities and I am curious as to the rationale behind using this instead of the normal PBS. The person who suggested this to me apparently got it off the internet and I am looking any relevant literature and/or valid reasons behind this before I try it out.

When, after macrophage isolation do you all count your cells. Currently I count the cells immediately after isolation and then seed them in the relevant flasks/plates, wait 3 hours then aspirate the non-adherent cells. It seems to me that it would be more logical, albeit more troublesome if I seed the cells, remove non-adherent cells, detach the adherent cells (PBS + 10mM glucose + 3mM EDTA) and then count them with turk's. What is the conventional practice for this?
Also, I allow 3 hours before aspirating non-adherent cells. Frankly I got this number by guesswork. How long should I wait?

I am stimulating cells with LPS to see the different cytokine production in WT and KO macrophages (with W. blot, RT-PCR, ELISA, FACS, etc). I read somewhere here that it is best to add LPS to media without FBS at all. Is this what I should be doing? If so, how do I go about doing it? Currently I am just adding LPS stock solution to the media (with 10%FBS) in the wells to the correct dilution. If I go without FBS, do I just seed the wells with cells in media without FBS or do I seed them with FBS and replace the media sans FBS just before adding LPS?

Thank you in advance. I would really appreciate any help.

hi spitsaliva

we isolate peritoneal exudates with ice cold incomplete media (media without FBS) and have no problem and i think PBS should work equally well.
if isolating macrophages from more than one mice than pool the exudate ,keep in ice , count and plate at desired density. we remove non adherent cells after 2 hours and it works well. i think there is no need to add more steps like detachment of the adherent cells (PBS + 10mM glucose + 3mM EDTA) etc because no of non adherent cells is almost same from every well of plate.
we add LPS to media with 10% FBS and get satisfactory results

good luck