माउस peritoneal गुहा कक्ष के… (Translated to Hindi)

Cite this Article: माउस peritoneal गुहा कक्ष के अलगाव

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

Abstract: माउस peritoneal गुहा कक्ष के अलगाव

peritoneal गुहा स्तनधारियों की एक झिल्ली ही सीमित है और तरल पदार्थ से भरे उदर गुहा, जो जिगर, तिल्ली, गैस्ट्रो आंत्र पथ और अन्य आंत का सबसे शामिल है. यह मैक्रोफेज, बी कोशिकाओं और टी कोशिकाओं प्रतिरक्षा कोशिकाओं का एक संख्या बंदरगाहों. peritoneal गुहा में भोले मैक्रोफेज के एक उच्च संख्या की उपस्थिति भोले ऊतक निवासी मैक्रोफेज (1) के संग्रह के लिए एक पसंदीदा स्थल बना देता है. peritoneal गुहा भी है एक अद्वितीय peritoneal गुहा निवासी बी सेल सबसेट पारंपरिक B2 कोशिकाओं को अतिरिक्त B1 कोशिकाओं के रूप में में जाना जाता है की उपस्थिति की वजह से बी कोशिकाओं के अध्ययन के लिए महत्वपूर्ण है. B1 कोशिकाओं B1a और B1b कोशिकाओं, जो CD11b और CD5 की सतह अभिव्यक्ति द्वारा प्रतिष्ठित किया जा सकता है में subdivided रहे हैं. B1 कोशिकाओं प्राकृतिक रोगज़नक़ों (2-4) की एक किस्म से जल्दी सुरक्षा प्रदान आईजीएम का एक महत्वपूर्ण स्रोत हैं. इन कोशिकाओं को प्रकृति (5) में autoreactive हैं, लेकिन वे कैसे autoimmunity को रोकने के लिए नियंत्रित कर रहे हैं अभी भी पूरी तरह से नहीं समझा जाता है. CD5, इसके विपरीत पर

Protocol: माउस peritoneal गुहा कक्ष के अलगाव

प्रक्रिया को शुरू करने से पहले, निम्नलिखित मदों एकत्र की है और तैयार होने की जरूरत है:
- बर्फ़
- 27G सुई के साथ 5ml सिरिंज
- 25g सुई के साथ 5ml सिरिंज
- स्टायरोफोम ब्लॉक और माउस बढ़ते के लिए पिंस
- ट्रे बढ़ते ब्लॉक जिस पर रखा जा सकता है
- कैंची और संदंश
- संग्रह ट्यूब
- 70% इथेनॉल
- पीबीएस के साथ 3% भ्रूण बछड़ा (FCS) सीरम (प्री ठंडा और बर्फ पर रखा)
माउस euthanize, यह 70% इथेनॉल के साथ स्प्रे और इसे अपनी पीठ पर स्टायरोफोम खंड पर माउंट.
एक कैंची और संदंश का प्रयोग peritoneum के बाहरी त्वचा में कटौती और धीरे इसे वापस खींचने के लिए भीतर peritoneal गुहा के अस्तर में त्वचा का पर्दाफाश.
Peritoneal गुहा में एक 27G सुई का उपयोग बर्फ के ठंडे पीबीएस (3% FCS के साथ) के 5 मिलीलीटर इंजेक्षन. सावधान पंचर करने के लिए नहीं जा रहा है किसी भी अंगों peritoneum में सुई धीरे पुश.
इंजेक्शन के बाद, धीरे पेरिटोनियम मालिश पीबीएस समाधान में किसी भी संलग्न कोशिकाओं को हटाना.
एक 25 ग्राम सुई बेवल, ऊपर सम्मिलित करें, peritoneum में 5 मिलीलीटर सिरिंज से जुड़ी है और तरल पदार्थ इकट्ठा जबकि सुई की नोक धीरे चलती वसा ऊतकों या अन्य अंगों द्वारा clogging से बचने के. संभव के रूप में अधिक तरल पदार्थ के रूप में ले लीजिए और सिरिंज से सुई को हटाने के बाद बर्फ पर रखा ट्यूबों में एकत्र सेल निलंबन जमा.
वैकल्पिक: दोहराएँ कदम 4-6
Peritoneum के भीतर की त्वचा में एक चीरा और जबकि एक संदंश के साथ त्वचा धारण एक प्लास्टिक पाश्चर पिपेट का उपयोग करने के लिए गुहा से शेष तरल पदार्थ इकट्ठा.
यदि चरण में 6 या 7 दिखाई रक्त संदूषण का पता चला है तब नमूना दूषित त्याग किया जाना चाहिए.
1500 RPM में 8 मिनट के लिए एकत्र सेल निलंबन, स्पिन सतह पर तैरनेवाला त्यागने और गिनती के लिए वांछित मीडिया या पीबीएस में कोशिकाओं resuspend.

वैकल्पिक प्रोटोकॉल thioglycollate प्राप्त मैक्रोफेज हासिल:

इस विधि मैक्रोफेज के एक उच्च उपज प्राप्त करने के लिए प्रयोग किया जाता है.

3% (w / v) प्रत्येक माउस के peritoneal गुहा में शराब बनानेवाला thioglycollate मध्यम (7) के 5 मिलीलीटर इंजेक्षन.
3-5 दिनों के लिए प्रतीक्षा करें, और कोशिकाओं के संग्रह के लिए 1 से ऊपर कदम आगे बढ़ना.

Nonelicited दृष्टिकोण की तुलना में लगभग 10 गुना अधिक मैक्रोफेज एकत्र किया जा सकता है. केवल चिंता का विषय है कि इस प्रक्रिया द्वारा प्राप्त मैक्रोफेज अपनी शारीरिक गुणों के कुछ अलग है.

प्रतिनिधि परिणाम:

एक unmanipulated माउस से 5-10 मिलियन peritoneal गुहा कोशिकाओं को एक अच्छा अलगाव प्रोटोकॉल के साथ प्राप्त किया जा सकता है. सभी जीवित कोशिकाओं के बीच 50-60% बी कोशिकाओं रहे हैं, ~ 30% मैक्रोफेज और 5-10% टी कोशिकाओं (चित्र 1) कर रहे हैं.

चित्रा 1. Peritoneal गुहा से अलग कक्षों की phenotype peritoneal गुहा वे कोशिकाओं के बाद अलगाव के साथ दाग थे विरोधी माउस TCRβ FITC, B220 पीई टेक्सास लाल, CD11b प्रशांत नीले CD23 पीई Cy7 और CD5 APC. प्रतिनिधि प्रवाह cytometric भूखंडों बी और टी कोशिकाओं (एक), (ख) मैक्रोफेज, B1 और B2 कोशिकाओं (ग) और B1a और B1b कोशिकाओं (घ) के प्रतिशत दिखा.

Discussion: माउस peritoneal गुहा कक्ष के अलगाव

Peritoneal गुहा कोशिकाओं के अलगाव अलग प्रतिरक्षा कोशिकाओं, मुख्य रूप से मैक्रोफेज और विशिष्ट बी सेल सबसेट के अध्ययन के लिए एक महत्वपूर्ण तकनीक है. हालांकि यह एक साधारण प्रक्रिया है, वहाँ कुछ महत्वपूर्ण कदम शामिल हैं. एकत्र नमूने में रक्त contaminating की उपस्थिति के लिए एक शुद्ध peritoneal गुहा सेल की आबादी को प्राप्त करने के लिए बचा जाना चाहिए. ग्रीवा अव्यवस्था द्वारा इच्छामृत्यु सावधानी से किया जाना चाहिए peritoneal गुहा में रक्त संक्रमण से बचने के लिए. वैकल्पिक रूप से सीओ ₂ इस्तेमाल किया जा सकता है . इसके अलावा, उचित देखभाल प्रक्रिया के दौरान peritoneal गुहा में puncturing मूत्राशय या किसी भी अन्य अंगों से बचने के लिए लिया जाना चाहिए. इंजेक्शन तरल पदार्थ का सबसे पुनर्प्राप्त भी सेल उपज के लिए महत्वपूर्ण है.

इस प्रक्रिया को व्यापक रूप से निवासी के मध्यम संख्या से बृहतभक्षककोशिका जीव विज्ञान का अध्ययन करने के लिए प्रयोग किया जाता है, unstimulated मैक्रोफेज peritoneal गुहा से आसानी से प्राप्त कर सकते हैं परिपक्व मैक्रोफेज में माइलॉयड पूर्वपुस्र्ष कोशिकाओं इन विट्रो में, फर्क बृहतभक्षककोशिका कॉलोनी उत्तेजक कारक (8 का उपयोग कर का श्रमसाध्य कार्य करने के लिए इसके विपरीत में, हो 9). peritoneal गुहा से बृहतभक्षककोशिका उपज thioglycollate निकालना पद्धति का उपयोग करके करके सुधार किया जा सकता है, हालांकि thioglycollate उपयोग मैक्रोफेज (7) के शारीरिक गुणों को बदल सकता है.

बी कोशिकाओं हमारी प्रतिरक्षा प्रणाली के दोनों सहज और प्रतिरक्षा अनुकूली में महत्वपूर्ण भूमिका निभा एक महत्वपूर्ण हिस्सा हैं. अलग बी सेल subpopulations के अलावा, B1 कोशिकाओं बी कोशिकाओं सहज रोगक्षमता, autoimmunity और प्रतिरक्षा विनियमन में शामिल की एक अद्वितीय सबसेट शामिल हैं. B1 कोशिकाओं को मुख्य रूप से peritoneal गुहा में स्थित हैं और स्वयं भरपाई कर रहे हैं. वे प्राकृतिक आईजीएम जो वायरस और बैक्टीरिया (10-12) की एक संख्या के खिलाफ सुरक्षा की पहली पंक्ति प्रदान करता है के प्राथमिक उत्पादकों में से एक हैं. B1 कोशिकाओं को भी 10-आईएल (6) है, जो एक महत्वपूर्ण प्रतिरक्षा विनियमन में शामिल साइटोकाइन है की एक प्रमुख स्रोत हैं. हालांकि कई अध्ययनों B1 कोशिकाओं के साथ जारी है, अभी भी उनकी विषम कार्यों के मूल्यांकन के लिए आगे गुंजाइश है, विशेष रूप से उनके नियामक भूमिका है. Peritoneal गुहा सेल अलगाव विधि अद्वितीय B1 कोशिकाओं के कार्यों का अध्ययन करने का अवसर प्रदान करता है.

Disclosures: माउस peritoneal गुहा कक्ष के अलगाव

Acknowledgements: माउस peritoneal गुहा कक्ष के अलगाव

इस काम के हिस्से में NIH अनुदान AI069358 और BloodCenter रिसर्च फाउंडेशन द्वारा समर्थित किया गया था.

Materials: माउस peritoneal गुहा कक्ष के अलगाव

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

References: माउस peritoneal गुहा कक्ष के अलगाव

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Mc Daniel, L.S., Benjamin, W.H., Forman, C. and Briles, D.E. Blood clearance by anti-phosphocholine antibodies as a mechanism of protection in experimental pneumococcal bacteremia. J. Immunol. 133, 3308-3312 (1984).
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: माउस peritoneal गुहा कक्ष के अलगाव

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

Automatic Translation

माउस peritoneal गुहा कक्ष के अलगाव

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