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Topic Started by sds
on 10/25/2007 18:07 PM
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Hi,
Does anyone know a method apart from doing FACS to isolate just the macrophages and not the others (neutrophils, B and T cells)? The simpler the better!:)
Thanks!
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Replies
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Posted By samm
on 10/26/2007 16:27 PM
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Adherence to plastic is the simplest procedure, and surprisingly effective for macs; density gradient centrifugation using Ficoll or Optiprep is good if you want it quicker (~15 mins as opposed to 2 h); enhanced density gradient using Ab-coated density beads for negative or positive selection takes longer (~35 mins), but gives very high purity, as does magnetic sorting using Miltenyi or similar MACS beads.
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Posted By samm
on 10/26/2007 16:30 PM
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Also, note that adherence to plastic will give you both macs and neutrophils - if you get PECS from a naive (unelicited/unactivated) mouse, you can expect to get ~3-6x10e5 macs; a thioglycollate elicited mouse will have 10-times the number.
Using ice-cold sucrose for flushing the PECS has worked best for me.
All the best!
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Posted By sds
on 10/26/2007 17:50 PM
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| Carson O Genic said: | | Adherence to plastic should work. Place the cells in 37°C tissue culture media (should contain at least 10% FBS) in a tissue culture flask in the incubator for about 2 hours. The monocyte/macrophages should stick to the plastic and the rest of the cells can be removed by gentle agitation of the flask to re-suspend the cells in the medium. |
Hi all, Thanks for your replies. I have been doing the way that u all suggested but just was wondering whether it is the right way to get the macrophages alone without any contamination from neutrophils. So Sam, if I use sucrose would that reduce neutrophils?
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Hi, it's interesting that you are trying to isolate monocytes, while I am trying to isolate neutrophils, but from rat peripheral blood.
Does anyone know the protocol?
-----Neutrophils isolation from rat peripheral blood?
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Posted By sds
on 10/30/2007 12:22 PM
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| Jason_Zhang said: | Hi, it's interesting that you are trying to isolate monocytes, while I am trying to isolate neutrophils, but from rat peripheral blood.
Does anyone know the protocol?
-----Neutrophils isolation from rat peripheral blood? |
Hi, Please refer this paper for neutrophil isolation in rats. Wang, J.P., Tsao, L.T., Raung, S.L., Lin, P.L. and Lin, C.N., 1999. Stimulation of respiratory burst by cyclocommunin in rat neutrophils is associated with the increase in cellular Ca2+ and protein kinase C activity. Free Radic. Biol. Med. 26, pp. 580588. I hope this helps! Good Luck!
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Posted By parvoman
on 10/31/2007 18:35 PM
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| Jason_Zhang said: | Hi, it's interesting that you are trying to isolate monocytes, while I am trying to isolate neutrophils, but from rat peripheral blood.
Does anyone know the protocol?
-----Neutrophils isolation from rat peripheral blood? |
I've done rat white blood cell preps which are essentially neutrophil preps. It involves using dextran (stock conc of 6%): Rat blood was collected in a tube containing 3.8% sodium citrate to prevent the blood from coagulating. Blood was mixed with dextran to a volume of 6ml and the final concentration of the dextran was 1.125%. This was carefully added on to the top of an equal volume of 1.35% dextran, in a 15mL tube and allowed to stand for 40mins on ice to separate the white blood cells from the red blood cells. The supernatant was removed and centrifuged at 220g for 5mins. The pellet was washed in cold PBS, centrifuged at 3000rpm for 5min and supernatant decanted. The resulting pellet was suspended in Tris-NH4Cl, incubated for 10mins on ice to allow for red blood cell lysis and centrifuged at 3000rpm for 5min at 40C. The resulting pelleted white blood cells were washed in ice cold PBS and centrifuged down at 3000rpm for 5min. This works pretty well.
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Posted By parvoman
on 10/31/2007 18:43 PM
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| sds said: | Hi,
Does anyone know a method apart from doing FACS to isolate just the macrophages and not the others (neutrophils, B and T cells)? The simpler the better!:)
Thanks! |
For isolation of macs from peritoneal lavages, we plate down on plastic in the absence of serum (in the presence of serum, B cells will stick). After 1 hour, we wash with ice cold PBS. This removes any B cells and neuts. If you are doing an assay the next day then you can wash again with warm PBS just in case any B cells or neuts remained after the ice cold PBS, since the next day they should be dead. You probably don't have to be too gentle with the washes. The macs re very difficult to remove from the plastic. If you are doing a phagocytosis assay with the macs then remember that macs that have eaten will become less adherent and could be more easily lost upon washing.
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Posted By sds
on 10/31/2007 11:27 AM
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| parvoman said: | | sds said: | Hi,
Does anyone know a method apart from doing FACS to isolate just the macrophages and not the others (neutrophils, B and T cells)? The simpler the better!:)
Thanks! |
For isolation of macs from peritoneal lavages, we plate down on plastic in the absence of serum (in the presence of serum, B cells will stick). After 1 hour, we wash with ice cold PBS. This removes any B cells and neuts. If you are doing an assay the next day then you can wash again with warm PBS just in case any B cells or neuts remained after the ice cold PBS, since the next day they should be dead.
You probably don't have to be too gentle with the washes. The macs re very difficult to remove from the plastic. If you are doing a phagocytosis assay with the macs then remember that macs that have eaten will become less adherent and could be more easily lost upon washing. |
Hi, Wouldn't the macs be affected without the serum for an hour? Is there any article that I can use as a reference? Thanks a ton!
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Hi all!!!
Does anyone know a protocol for isiolation of neutrophils from the peritoneum?
I extract the cells by injection of thioglycolate 2.9% into the peritoneum and after 4 hours draw the cells. By using this protocol I get a mixture of neutrophils and monocytes. I want to seperate between the two. How is that done?
George
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Posted By samm
on 2/14/2008 21:10 PM
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If you want to accurately differentiate between monocytes and neutrophils in suspension, you can do it by FACS. Differential cell counts based on staining and nuclear morphology is another way.
For FACS, using 7/4 and Ly6G Abs is a good way to differentiate the two.
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Posted By gizem
on 10/15/2008 2:06 AM
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I have problems with white blood cells after I collect peritoneal macrophages. I am injecting 4% thioglycollate ip and collect cells from peritoneum after 4 days. I could not get rid of round cells I also saw one macrophage was eating. I am checking proinflammatory cytokine and it gives me false response I think. What do you recommend? You are using serum free for the first hour only them after removing the other cells so you use medium with serum?
thank you
good luck
| sds said: | | parvoman said: | | sds said: | Hi,
Does anyone know a method apart from doing FACS to isolate just the macrophages and not the others (neutrophils, B and T cells)? The simpler the better!:)
Thanks! |
For isolation of macs from peritoneal lavages, we plate down on plastic in the absence of serum (in the presence of serum, B cells will stick). After 1 hour, we wash with ice cold PBS. This removes any B cells and neuts. If you are doing an assay the next day then you can wash again with warm PBS just in case any B cells or neuts remained after the ice cold PBS, since the next day they should be dead.
You probably don't have to be too gentle with the washes. The macs re very difficult to remove from the plastic. If you are doing a phagocytosis assay with the macs then remember that macs that have eaten will become less adherent and could be more easily lost upon washing. |
Hi,
Wouldn't the macs be affected without the serum for an hour?
Is there any article that I can use as a reference?
Thanks a ton! |
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Posted By rlsun
on 10/22/2008 8:10 AM
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| samm said: | Also, note that adherence to plastic will give you both macs and neutrophils - if you get PECS from a naive (unelicited/unactivated) mouse, you can expect to get ~3-6x10e5 macs; a thioglycollate elicited mouse will have 10-times the number.
Using ice-cold sucrose for flushing the PECS has worked best for me.
All the best! |
Hi, I want to know why thioglycollate can elicit mouse ,another question,someone elicit mouse with thioglycolate broth ,,which one i should select ,thioglycolate or thioglycolate broth ? Thank you
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Posted By samm
on 10/22/2008 14:34 PM
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Aged 4% thioglycollate in saline works very well. Ready made formulations tend to differ, but the simple recipe with Sigma thioglycollate has worked just fine. The 'elicited' macrophages differ from 'native' ones, but they do not have an 'activated' macrophage phenotype.
The aging part can be done by storing the autoclaved sterile thioglycollate in the refrigerator over several weeks (2-3 atleast), preferably several months. I'm not absolutely sure why the aged version is better, but theres years of anecdotal data from labs all over the world on this one!
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