小鼠腹膜腔细胞的分离 (Translated to Chinese)

Cite this Article: 小鼠腹膜腔细胞的分离

Ray, A., Dittel, B. N. Isolation of Mouse Peritoneal Cavity Cells. J. Vis. Exp. (35), e1488, doi:10.3791/1488 (2010).

腹膜腔是腹腔膜结合的充满液体的哺乳动物，其中包含的肝，脾，大部分的胃肠道和其他脏器。港口包括巨噬细胞，B细胞和T细胞的免疫细胞的数量。一个天真腹腔巨噬细胞大量存在，使一个天真的组织驻地巨噬细胞（1）收集的首选网站。腹膜腔是因为存在一个独特的腹腔居民的B细胞为B1细胞除了常规B2细胞的一个子集B细胞的研究也很重要。 B1细胞分为菌素和B1B细胞，它可以通过表面的CD11b和CD5表达尊敬。 B1单元格是一个由多种病原体（2-4）早期保护自然提供的IgM的重要来源。这些细胞是自身反应（5）性质，但它们是如何控制，以防止自身免疫性疾病仍然是不完全理解。相反，CD5

Protocol: 小鼠腹膜腔细胞的分离

开始之前的过程中，下列项目需要收集和准备：
- 冰
- 27克针5ml注射器
- 25克针5ml注射器
- 安装鼠标的泡沫塑料块和引脚
- 可以放在托盘上安装块
- 剪刀和镊子
- 收集管
- 70％的乙醇
- PBS与3％小牛血清（FCS）（前冷冻保存在冰）
安乐死的鼠标，喷以70％乙醇和安装在它的后面的发泡胶块。
使用剪刀和镊子切开腹膜外的皮肤，轻轻一拉，内层衬里的腹腔暴露。
使用27克针进入腹膜腔注入5毫升冰冷的PBS（含3％FCS）。慢慢推针腹膜穿刺小心，不要被任何机关。
注射后，轻轻按摩腹膜驱逐任何贴壁细胞的PBS液。
插入一个25克的针，锥，附着在腹膜5 ml注射器和搜集流体移动时针尖轻轻地避免脂肪组织或其他器官堵塞。尽可能收集尽可能多的液体和存款取出后从注射器针管放在冰上收集细胞悬液。
可选：重复步骤4-6
请在腹膜内的皮肤切口，并与钳皮肤而使用塑料的巴斯德吸管收集剩余的液体从腔。
如果在步骤6或7可见血液污染检测，然后被污染的样品应该被丢弃。
收集的细胞悬液在1500转的旋转，8分钟，弃去上清液，并计算所需的媒体或PBS重悬细胞。

替代协议获得硫乙醇酸盐引起巨噬细胞：

使用此方法，以获得更高的巨噬细胞的产量。

3％（W / V）布鲁尔硫乙醇酸盐培养基（7）到每只小鼠腹腔注射5毫升。
等待3-5天，并继续执行步骤1段收集细胞。

nonelicited方法相比，可以收集到约10倍多的巨噬细胞。唯一担心的是，此过程中获得的巨噬细胞，在他们的一些生理特性不同。

代表性的成果：

从unmanipulated鼠标，5-10万元的腹腔细胞，可以得到具有良好的隔离协议。在所有活细胞，B细胞的50-60％，〜30％的巨噬细胞和5-10％的T细胞（图1）。

图1。从腹膜腔中分离的细胞表型。以下隔离腹腔细胞，他们沾满抗鼠TCRβ- FITC，B220 - PE德州红，CD11b的太平洋蓝，CD23 - PE - Cy7和CD5 - APC。代表流式细胞仪图显示B细胞和T细胞（A），巨噬细胞（B），B1和B2的细胞（C）和阿维菌素B1a和B1B细胞（D）的百分比。

Discussion: 小鼠腹膜腔细胞的分离

腹腔细胞的分离，不同的免疫细胞，主要是巨噬细胞和特定的B细胞亚群的研究是一个重要的技术。虽然这是一个简单的过程，也有一些涉及的关键步骤。应避免污染所收集的样品中血液的存在，获得纯腹腔细胞群。颈椎脱位安乐死应做仔细，以避免在腹腔的血液污染。或者可以使用的CO _2。此外，在手术过程中适当的照顾中应采取，以避免在腹腔穿刺膀胱或任何其他机关。注入流体恢复大部分也是很重要的细胞产量。

此过程被广泛用于研究自中等数量的居民巨噬细胞生物学，未刺激巨噬细胞可以很容易地获得从腹腔，在髓系祖细胞在体外分化成成熟的巨噬细胞，巨噬细胞集落刺激因子（8费力的任务，9）。从腹腔巨噬细胞的产量可以提高使用硫乙醇酸盐启发式，虽然硫乙醇酸盐的使用可能会改变的巨噬细胞的生理特性（7）。

B细胞是先天和适应性免疫中扮演关键角色，我们的免疫系统的重要组成部分。在不同的B细胞亚群，B1细胞构成独特的先天免疫，自身免疫和免疫调节B细胞参与的一个子集。 B1细胞主要位于腹腔和自我补充。它们是自然的IgM它提供了针对病毒和细菌（10-12）保护的第一线的主要生产商之一。 B1细胞的IL - 10（6），这是一个重要的细胞因子参与免疫调节的主要来源。虽然几项研究已经与B1单元格的追求，仍然有余地为进一步评价其对比功能，特别是其监管作用。腹腔细胞的分离方法研究B1细胞的功能提供了独特的机会。

Disclosures: 小鼠腹膜腔细胞的分离

Acknowledgements: 小鼠腹膜腔细胞的分离

这项工作是支持部分由美国国立卫生研究院授予AI069358和BloodCenter研究基金会。

Materials: 小鼠腹膜腔细胞的分离

Name	Company	Catalog Number	Comments
Thioglycollate Medium, Brewer Modified	BD Biosciences	211716

References: 小鼠腹膜腔细胞的分离

Zhang, X., Goncalves, R. and Mosser, D.M. The isolation and characterization of murine macrophages. Current Protocols in Immunology (2008).
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Paciorkowski, N., Porte, P., Shultz, L.D. and Rajan, T.V. B1 B lymphocytes play a critical role in host protection against lymphatic filarial parasites. J. Exp. Med. 191, 731-736 (2000).
Mercolino, T.J., Arnold, L.W., Hawkins, L.A. and Haughton, G. Normal mouse peritoneum contains a large population of Ly-1+ (CD5) B cells that recognize phosphatidyl choline: relationship to cells that secrete hemolytic antibody specific for autologous erythrocytes. J. Exp. Med. 168, 687-698 (1988).
O'Garra, A., Chang, R., Go, N., Hastings, R., Haughton, G. and Howard, M. Ly-1 B (B-1) cells are the main source of B cell derived interleukin 10. Eur. J. Immunol. 22, 711-717 (1992).
Hoover, D.L. and Nacy, C.A. Macrophage activation to kill Leishmania tropica: Defective intracellular killing of amastigotes by macrophages elicited with sterile inflammatory agents. J. Immunol. 132, 1487-1491 (1984).
Austin, P.E., McCulloch, E.A. and Till, J.E. Characterization of the factor in L-cell conditioned medium capable of stimulating colony formation by mouse marrow cells in culture. J. Cell Physiol. 77, 121-134 (1971).
Stanley, E.R. The macrophage colony-stimulating factor, CSF-1. Methods Enzymol. 116, 564-587 (1985).
Baumgarth, N., Chen, J., Herman, O.C., Jager, G.C. and Herzenberg, L.A. The role of B-1 and B-2 cells in immune protection from influenza virus infection. Curr. Top. Microbiol. Immunol. 252, 163-169 (2000).
Baumgarth, N., Herman, O.C., Jager, G.C., Brown, L.E., Herzenberg, L.A. and Chen, J. B-1 and B-2 cell-derived immunoglobulin M antibodies are nonredundant components of the protective response to influenza virus infection. J. Exp. Med. 192, 271-280 (2000).
Berland, R. and Wortis, H.H. Origins and functions of B-1 cells with notes on the role of CD5. Annu. Rev. Immunol. 20, 253-300 (2002).

Ask the Author: 小鼠腹膜腔细胞的分离

29 Comments

Hello Dr. Ray,
Thank you for your beautiful demo & the theory.
I have 2 questions:
1. How can one differentiate between the B & T-cells & macrophages without using FACS ?
2. Is there a way to get only the macrophages ?
Thanks.
Chandra

Posted by: ChandraMarch 10, 2010, 8:29 AM

1.FACS buffer is just PBS with 2%FBS. Actually, you can only use PBS for isolating the peritoneal cells. However, I don't understand what you are asking.
2. You can get macrophages by letting cells adhere to the plate for 2 hours, and wash the cells with PBS 2 times. Since macrophages are the only cells that can adhere to the plate, other cells are gonna be washed off.

1.1

Posted by: Shan Y.May 5, 2010, 8:38 PM

Dear Chandra:
You can use magnetic cell sorting to obtain the different cell populations post isolation from the peritoneal cavity and through thioglycollate elicitation you will obtain more macrophages. You can purify the macrophages either by adhering them to petri dishes or through cell sorting.
Thanks,
Avijit

1.2

Posted by: Bonnie D.June 8, 2010, 5:13 PM

Beautiful tutorial! Thank you!

Posted by: AnonymousApril 29, 2010, 6:00 PM

Thanks

2.1

Posted by: Bonnie D.June 8, 2010, 5:01 PM

Thank you. Great job.
How would you store the isolated cells and for how long?

Posted by: AnonymousMay 1, 2010, 7:08 AM

These are primary cells and we use them for phenotypic analysis or culture them immediately after isolation.

3.1

Posted by: Bonnie D.June 8, 2010, 5:00 PM

Dear Dr. Ray,

My PI was wondering where you purchased the antibodies used for your figures. Thanks for the demo!
Thanks so much,

-Mike

Posted by: MikeJune 8, 2010, 11:26 AM

Dear Mike:
The antibodies were obtained from ebioscience and BD Biosciences.
B220 (BD Biosciences); TCR beta (ebioscience); CD5 (BD Biosciences); CD11b (ebioscience); CD23 (ebioscience).
Thanks,

Avijit

4.1

Posted by: Bonnie D.June 8, 2010, 4:49 PM

Thanks so much!

4.1.1

Posted by: MikeJune 9, 2010, 4:42 PM

Dear Dr. Ray,

Thank you for this great video!

I am isolating macrophages from the periotoneal cavity the same way you do it. But after a week, when I do Inmunocitochemistry I found Desmin+/ smooth muscle+ cells... and I am wondering why?? other cells could be contaminating my cultures???
May I fail during the wash step and not removing all the non-adherent cells??

Other questions is, how long do this cells live in culture??

I will apreciatte your help!

Diana.

Posted by: DIANA S.July 12, 2010, 9:38 AM

Dear Dr.Avijit Ray,
My name is Salman Khan and Im PhD course student in South Korea. Currently, Im working on primary culturing of macrophages for the measurment of nitric oxide and PGE2 production in the culture media. For my experiment, I followed all your discribed procedure for the isolation of peritonial macrophages and seeded in 24 well plate, and incubate for two hours in 37C. I treat the cells with my samples and stimulated with LPS (1ug/ml) for 20 h. When, I cheched the nitric oxide concentration (using griess reagent method), but there was no LPS stimulation. would you mind to give me some propoer and nice suggestion??
I will be realy grateful to you.
Thank you so much.

Salman

Posted by: AnonymousSeptember 14, 2010, 2:54 AM

Dear Mr. Salman Khan:
My suggestion would be to culture cells at a concentration of 1 million cells per ml in 96 well flat-bottom microtiter plate and stimulate them with 5-10 ug/ml LPS for 24h. Post culture centrifuge the samples and collect cell free supernatant. Aliquot 50ul of this supernatant from each sample in a 96 well plate and add equal amount of Griess reagent to each sample. Incubate for 10 min at room temperature and measure absorbance.

Thanks,

Avijit Ray

6.1

Posted by: Bonnie D.September 14, 2010, 2:50 PM

Dear Dr. Avijit Ray
my name is Jameel, I am a Ph.D student in Jordan, currently I am working on murine peritoneal macrophages for the measurement IL-6, TNF-alpha and IL1 Beta after stimulation with LPS , what is the best LPS concentration to be used and the best cell density / well (96 well plate) and the incubation time. Second question is can I use peritoneal macrophages to measure PIP3 level by ELISA after stimulatin with LPS and with what conditions, thank you

6.1.1

Posted by: JameelJanuary 3, 2011, 9:21 AM

Posted by: AnonymousSeptember 14, 2010, 4:02 AM

Posted by: AnonymousSeptember 14, 2010, 5:02 AM

Dr.Ray,
Thank for you nice suggestion previously.
Now my question is??
After 2 h incubation of primary cells in 24 well plate I treated with my sample and incubate for 2h. Then, stimulated with 10 ug/ml LPS for 20h, and 20h and 24h. I determined NO assay after 20 h, and 24h incubations after LPS stimulation, but, no stimulation with LPS were observed. While I used DMEM media and balb c mouse.
Would you mind to give me proper suggestion in order to proceed my experiments?
Thank you so much.
Salman

Posted by: Salman KhanSeptember 22, 2010, 5:29 AM

First of all, Balc mice are much less responsive to LPS than C57B6. Second, 10 ug/mL LPS is a VERY high concentration, and 20 hr is a VERY long stimulation. In my hand, I can get a very robust response with 1 ng/mL LPS. In term of serected cytokine levels, black 6 macrophage secreted 3 times more than Balbc ones. I would suggest you try to run a time course first to determine the optimal time point for medium collection.

9.1

Posted by: Zhifei S.September 22, 2012, 11:53 PM

Dear Dr. Ray,

My name is Branislav and I am a PhD student working
in an immunology lab in Jena, Germany. We are also focused
on different B cells populations including B1 cells and Marginal zone B cells.
My current aim is to perform a peritoneal B cell transfer from one mouse to another,
and for this I would like to use magnetic negative selection, depletion of all non-B cells
in the peritoneal wash. What markers would you use to effectively remove all T cells and macrophages
from the peritoneal exudate? I guess TCRab would be enough for T cells? And what about periotoneal macrophages,
do they have any specific markers, different from B1 cells?

Thank you very much in advance,
I am looking forward to your answer.
Best regards,

Branislav
Fritz Lipmann Institute
Jena, Germany

Posted by: Branislav K.October 28, 2010, 5:00 PM

Dear Dr. Ray, it is a wonderful presentation. Thank you very much indeed. I am working in Mexico City. I want to isolate peritoneal macrophages and know how many type 2 and typ2 macrophages I have after producing surgical peritoneal adhesion (time course after surgery). I would like to know if I can also take some of the aspirated liquid to measure interleukins 1, 6 and 10 (same animal). May I measure interleukins in the centrifuge supernatant? can I do immunohistochemistry of the isolated macrophages? Thank you very much and please receive my best regards from Mexico

Posted by: Cleva VillanuevaNovember 2, 2010, 2:38 PM

Hi, do you have a similar video for isolation of primary sinovial fibroblasts from mouse joints ??!!

Posted by: AnonymousDecember 4, 2010, 7:07 AM

I am sorry, but we do not have a prootcol for the isolation of joint fibroblasts.

12.1

Posted by: AnonymousDecember 6, 2010, 10:42 AM

Dear Dr. Ray, I want to isolate mouse peritoneal macrophages to check some signaling pathway. Can you tell me how to make 3% brewer thioglycollate medium?? what is its solvent?? Waitng for kind reply.

Posted by: Md. Jamal UddinApril 19, 2011, 10:31 PM

Dear Jamal,

You can follow the method as mentioned in ref # 7.

Thanks,

Avijit

13.1

Posted by: Avijit RayJune 19, 2011, 5:05 PM

Dear Dr. Ray,

Thank you for this great video!
We previously make it in dd water after boiling and autoclave. But the peritoneal macrophage cells are conglobated. I'm not sure whether we had something wrong in making the 3% (w/v) Brewer thioglycollate medium. Do you have any suggestions for us?

Thank you very much!

Ivy

Posted by: IvyJune 13, 2011, 4:19 AM

Dear Ivy,

Dissolve thioglycollate medium in DD water and autoclave it.
It is not uncommon to have some globular macrophages. Did you check by flow cytometric staining whether the conglobated cells as obtained by you are macrophages? You may get some neutrophils and lymphocytes depending on the time between thioglycollate injection and collection of cells. Collect PerC cells 3-4 days after thioglycollate injection to obtain optimal yield of macrophages.

Thanks,

Avijit

14.1

Posted by: Avijit RayJune 19, 2011, 5:48 PM

Hello, all!
I am very new in this field, and please tell me
1. Why it takes 3-5 days after injection thioglycollate medium? May I collect peritoneal fluid directly after injection thioglycollate medium same as PBS?
2. What does the mean "macrophages obtained by this procedure differ in some of their physiological properties."?

Posted by: dashaJune 17, 2011, 2:12 AM

Dear Dasha,

Thioglycollate medium is not used for collecting cells unlike PBS. It is used to elicit macrophages to obtain better yield and 3-4 days is the optimal time. You can collect peritoneal macrophages without using thioglycollate. Only the yield will be less.
Check ref # 7 for altered macrophage properties using thioglycollate.

Thanks,

Avijit

15.1

Posted by: Avijit RayJune 19, 2011, 5:57 PM

hello!!
can we use 1%starch instead of thioglycollate?? if ;yes which one is preferable?/

Posted by: Meena KusiJuly 19, 2011, 5:48 AM

Hi,

Starch can be used but I prefer thioglycollate.

Thanks,

Avijit

Posted by: Avijit RayJuly 19, 2011, 10:28 AM

Hi,

Great video! How long can we keep these cells in culture for, and how often do you feed/passage them?

Thanks,

Stefanny

Posted by: stefannySeptember 14, 2011, 3:50 PM

Hi Stefanny,

The macrophages can be can be kept in culture using medium containing colony stimulating factors (CSFs). Media should be changed depending on the viability of the cells. Without CSFs the cells can be cultured for 48-72 hrs.

Thanks,

Avijit

18.1

Posted by: Avijit RaySeptember 18, 2011, 3:52 PM

Respected Sir,
I am Kiran Kundu. Now I am a project assistant at National Brain Research Centre, India. I am working upon Japanese Encephalitis using peritoneal macrophage. But during isolation of peritoneal macrophage from 3 to 4 weeks mice yield or number of cells is very low.So I want your suggestion to get a good result.
Yours Obediently
Kiran Kundu

Posted by: Kiran K.February 8, 2012, 11:50 AM

Dear Kiran,

Macrophage yeild from peritoneal cavity varies with mouse strain, age and sex. You may obtain more cells using 6-8 weeks old mice. Perform steps 4-6 twice and try to collect most of the fluid from the peritoneal cavity.

Thanks,

Avijit

19.1

Posted by: Avijit RayFebruary 19, 2012, 1:20 PM

Dear Dr. Avijit
I am researching on macrophages are obtained from peritoneal cavity in mouse. According to your presentation, I have some question;
Can I mix 3-5 fluid together and make a pool of them?
Can I use CD14 for detecting of macrophages of mouse; I mean CD 14 is specific to human?
Can I treated on mouse in vivo and investigate morphology and immunology parameter in vitro? i mean they may keep their changes in vitro as clear as in vivo?
How can I stain them? Which of these? Trypan Blue or Giemsa or Hematoxylin?
Sincerely
Latifeh,IRAN

Posted by: latifehFebruary 19, 2012, 8:56 AM

Dear Latifeh,

Do you mean mixing cells from 3-5 mice? If so, depending on your experimental design you can.
Mouse cells express CD14. I would suggest using CD14 in combination with CD11b and B220 (to gate out B cells) for the detection of peritoneal cavity macrophages.
You can treat mice in vivo and isolate PerC macrophages to study immune parameters depending on your experimental design.
Use Trypan blue to determine cell viability and use Wright-Giemsa staining for morphological studies.

Thanks,

Avijit

20.1

Posted by: Avijit RayFebruary 19, 2012, 1:48 PM

Is it possible to lyse the red blood cells in your cell preparation if you do get some contamination? I use ACK lysis buffer when isolating spleen cells for this same purpose. Thank you for your video and helpful advice!

Posted by: MorrisMarch 1, 2012, 4:37 PM

Dear Morris,

It is possible to lyse RBCs using ACK lysis buffer, but you cant get rid of the contaminating WBCs and you will obtain a mixed population of cells from peritoneal cavity and blood.

Thanks,

Avijit

21.1

Posted by: Avijit RayMarch 2, 2012, 5:51 PM

Dear Dr. Avijit
how can i get rid of the contaminating WBCs?

Posted by: latifehMarch 7, 2012, 5:08 AM

Dear Latifeh,

You can't get rid of the contaminating blood WBCs. If pure PerC cell population is required discard any sample that is contaminated with blood.

Thanks,

Avijit

Posted by: Avijit RayMarch 7, 2012, 11:37 AM

Dear Dr. Ray,

I am isolating macrophages from the periotoneal cavity the same way you do it. I have put cell 2 hours in incubator and washed with PBS. When I saw plate, many cells were attached to plate. Then I centrifuged at 1700 rpm 10 min, but I almost could not get pellet...pellet was very less and on the wall only...Please tell me what I did wrong?

Posted by: dashaMarch 27, 2012, 4:51 AM

Dear Dasha,

Post incubation, discard the non-adherent cells and gently wash the petri dish with PBS (PBS should be at room temperature). Harvest the adhered cells from the petri dish using a cell scraper and cold PBS. Repeat the process once and centrifuge the collected cells in a polypropylene tube.

Thanks,

Avijit

Posted by: Avijit RayMarch 27, 2012, 4:39 PM

Dear Dr.Ray,

Thanks for the great video. Should the isolation of peritoneal cells be done in sterile condition (i.e. laminar hood)? Thanks.

Posted by: shien l.February 26, 2013, 8:59 AM

Hi Shien,

If you want to use the isolated peritoneal cavity cells for in vitro cell culture, the procedure should be performed under sterile conditions.

Thanks,

Avijit

26.1

Posted by: AnonymousFebruary 28, 2013, 1:02 PM

Dear Dr.Ray,

Thanks for helping me to improve my mouse peritoneal macrophages isolation.
I have an issue that i would like to discuss with you.

1)Do you simply distinguish the different cell populations by adherence? Is it trustable that B and T cells do not adhere to the plates?

2)Can you distinguish between the different macrophage populations, that is, do you know if is possible to assess which type of macrophage is present? I know there are CD markers characteristic of each macrophage, but i read that they are not specific..

Thank you very much!
Sincerely

Posted by: In�s S.March 20, 2013, 9:18 AM

An enriched macrophage population (90-95%) is obtained by adherence to petri dishes, which can be further sorted to attain a higher purity.

You can use CD markers to distinguish the different macrophage populations.

Thanks,

Avijit

27.1

Posted by: AnonymousApril 1, 2013, 2:11 PM

An enriched macrophage population (90-95%) is obtained by adherence to petri dishes, which can further sorted to attain a higher purity.

You can use CD markers to distinguish the different macrophage populations.

Thanks,

Avijit

Posted by: AnonymousApril 1, 2013, 2:03 PM

Dear Dr Ray,

When i isolate peritoneal macrophages, i immediately plaque 200x10^3 cells, but it is a bit difficult to distinguish and therefore count macrophages at microscope. On microscope i observe two different populations: big and small spheres. I consider that the big spheres are macrophages. So, only after count the cells and plaque them, i am able to observe their adherence. My doubt on this procedure is, how can i ensure that the cells i am counting are macrophages? I know that after 1 day, they adhere to the plaque, but the number may not be right.

Thank you very much!

Posted by: In�s S.April 2, 2013, 5:38 AM

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Date Published	1/28/2010
JoVE Section	Immunology and Infection
JoVE Issue	35
Comments	29 Comments
View Count	Loading... (Details…)
DOI	doi:10.3791/1488

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小鼠腹膜腔细胞的分离

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