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 JoVE Immunology and Infection

Fare Periton Boşluk Hücreleri İzolasyon

1, 1

1BloodCenter of Wisconsin, Blood Research Institute

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    Summary

    Periton boşluğuna memelilerde doğuştan gelen bağışıklık yanıtları için çok önemli olan farklı bağışıklık hücre popülasyonlarının içerir. Verimli bir izolasyon yöntemi, bu hücrelerin biyokimyasal ve fonksiyonel analizler için gereklidir. Burada fare periton boşluğuna hücrelerin izolasyonu için kapsamlı bir yöntem sağlar.

    Date Published: 1/28/2010, Issue 35; doi: 10.3791/1488

    Cite this Article

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

    Abstract

    Periton boşluğuna memelilerin karaciğer, dalak, mide-bağırsak ve diğer iç organları içeren bir membran-bağlı ve karın boşluğunda sıvı dolu. Bu bağışıklık hücreleri, makrofajlar, B hücreleri ve T hücreleri de dahil olmak üzere bir dizi barındırıyor. Periton boşluğunda naif makrofajlar yüksek sayıda varlığı naif doku ikamet eden makrofaj (1) toplanması için tercih edilen bir site yapar. Peritoneal kavite varlığı nedeniyle geleneksel B2 hücrelerine ek olarak B1 hücreler olarak bilinen eşsiz bir periton boşluğuna yerleşik B hücre alt B hücreleri çalışma için önemlidir. B1 hücreleri CD11b ve CD5 yüzey ifadesi ile ayırt edilebilir B1a ve B1b hücreleri, bölünmüştür. B1 hücreleri çeşitli patojenler (2-4) bir erken koruma sağlayan doğal IgM önemli bir kaynak. Bu hücreler, Doğa (5) otoreaktif, ama onlar otoimmünite önlemek için nasıl kontrol edilir hala tam olarak anlaşılmış değildir. Aksine, CD5

    Protocol

    1. Işlemine başlamadan önce, aşağıdaki öğeleri toplanmış ve hazırlanmış olması gerekir:
      • Buz
      • 27g iğne ile 5ml şırınga
      • 25g iğne ile 5ml şırınga
      • Strafor blok ve fare montaj pimleri
      • Montaj bloğu yerleştirilmiş olabilir Tepsi
      • Makas ve forseps
      • Toplama tüpleri
      • % 70 etanol
      • PBS ile% 3 fetal buzağı serumu (FCS) (Ön buz üzerinde soğutulmuş ve muhafaza)
    2. Fare Euthanize,% 70 etanol ile sprey ve sırtında strafor blok üzerine monte edin.
    3. Bir makas ve forseps kullanarak periton dış deri kesme ve periton boşluğuna kaplayan iç deri hafifçe ortaya çıkarmak için geri çekin.
    4. 27g bir iğne kullanılarak peritoneal kavite içine buz PBS (% 3 FCS) 5 ml enjekte edilir. Herhangi bir organ dikkatli olmazsanız ponksiyon için periton iğne yavaşça itin.
    5. Enjeksiyondan sonra, periton PBS çözümü herhangi bir bağlı hücreleri çıkarmak için hafifçe masaj yapın.
    6. Periton bir 5 ml şırınga bağlı olarak, 25 g iğne, konik takın ve sıvı yağ dokusu veya diğer organlar tarafından tıkanmasını önlemek için iğne ucu hafifçe hareket ettirerek toplamak. Mümkün olduğunca fazla sıvı olarak toplayın ve şırınga iğnesi çıkardıktan sonra buz üzerinde tutulmalıdır tüplerde toplanan hücre süspansiyonu yatırmak.
      İsteğe Bağlı: Tekrar adım 4-6
    7. Periton iç deride bir kesi yapın ve bir forseps ile cilt tutarken boşluğunda kalan sıvı toplamak için plastik bir Pasteur pipeti kullanın.
    8. 6 ya da 7 adımda görünür kan kontaminasyonu tespit edilirse kontamine örnek atılmalıdır.
    9. 1500 RPM, 8 dakika boyunca toplanan hücre süspansiyonu Spin süpernatantı atmak ve istenen ortam veya PBS içinde hücre sayımı için tekrar süspansiyon haline getirin.

    Tiyoglikolat elde Alternatif protokol makrofajlar ortaya çıkardı:

    Bu yöntem, makrofajların daha yüksek verim elde etmek için kullanılır.

    1. % 3 (w / v) her fare periton boşluğuna Brewer tiyoglikolat orta (7) 5 ml enjekte edilir.
    2. 3-5 gün bekleyin ve hücrelerin toplanması için yukarıdaki 1. adımı geçin.

    Nonelicited yaklaşımı ile karşılaştırıldığında, yaklaşık 10 kat daha fazla makrofaj toplanabilir. Tek endişe, bu yordamı ile elde edilen makrofajlar bazı fizyolojik özellikleri farklı olduğunu.

    Temsilcisi sonuçları:

    Unmanipulated fare, 5-10 milyon periton boşluğuna hücreleri iyi bir izolasyon protokolü ile elde edilebilir. Tüm canlı hücreler arasında,% 50-60 B hücreleri, ~% 30 makrofajlar ve T hücreleri (Şekil 1)% 5-10.

    Şekil 1
    Şekil 1. Periton boşluğuna izole hücre fenotipi periton boşluğuna hücreleri takiben izolasyonu ile boyandı anti-fare TCRβ FITC, B220-PE-Texas kırmızı, CD11b-Pasifik mavi, CD23-PE-Cy7 ve CD5-APC. Temsilci akış sitometrik araziler, B ve T hücreleri (a), makrofajlar (b), B1 ve B2 hücreleri (c) ve B1a ve B1b hücreleri (d) yüzdeleri göstermektedir.

    Discussion

    Periton boşluğuna hücrelerinin izolasyonu, farklı bağışıklık hücreleri, başta makrofajlar ve spesifik B hücre alt çalışma için önemli bir tekniktir. Bu basit bir süreç olmasına rağmen, bazı kritik adımlar vardır. Saf bir periton boşluğuna hücre popülasyonu elde etmek için toplanan örnek kan kirlenmesine varlığı kaçınılmalıdır. Servikal dislokasyon tarafından Ötenazi periton boşluğunda kan kirlenmesini önlemek için dikkatli bir şekilde yapılmalıdır. Alternatif olarak CO 2 kullanılabilir. Buna ek olarak, prosedüre uygun bakım sırasında peritoneal kavite, mesane ya da herhangi bir diğer organlara delinmesiyle önlemek için alınmalıdır. Enjekte edilen sıvı en kurtarma, hücre verimi önemlidir.

    Bu prosedürü, yaygın olarak orta numaraları bu yana ikamet eden makrofaj biyoloji okumak için kullanılır, unstimulated makrofajlar kolayca makrofaj koloni stimüle edici faktör (8 kullanılarak, in vitro olgun makrofajlara miyeloid progenitör hücrelerin ayırt zahmetli görev aksine, periton boşluğuna elde edilebilir , 9). Tiyoglikolat kullanımı makrofajlar (7) fizyolojik özelliklerini değiştirebilir olsa da periton boşluğunda makrofaj verim, tiyoglikolat ortaya çıkarma yöntemi kullanarak geliştirilebilir.

    B hücreleri bağışıklık sisteminin doğal ve adaptif bağışıklık hem de önemli rol oynayan önemli bir parçasıdır. Farklı B hücre alt arasında, B1 hücreleri B hücreleri doğuştan gelen bağışıklık, otoimmünite ve bağışıklık düzenleme yer eşsiz bir alt oluşmaktadır. B1 hücreler öncelikle periton boşluğunda bulunan ve kendini yeniler. Onlar, ilk satırı, bir virüs sayısı ve bakteriler (10-12) karşı koruma sağlar doğal IgM birincil üreticiler biri. B1 hücreler de immün regülasyon yer alan önemli bir sitokin olan IL-10 (6), önemli bir kaynağıdır. B1 hücreleri ile çeşitli çalışmalar takip olmasına rağmen, hala özel, kendi karşıt fonksiyonları daha fazla değerlendirme için düzenleyici rol kapsamı vardır. Periton boşluğuna hücre izolasyon yöntemi B1 hücrelerinin fonksiyonlarını incelemek için eşsiz bir fırsat sunuyor.

    Disclosures

    Acknowledgements

    Bu çalışma NIH hibe AI069358 ve BloodCenter Araştırma Vakfı tarafından kısmen desteklenmiştir.

    Materials

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

    References

    1. Zhang, X., Goncalves, R. and Mosser, D.M. The isolation and characterization of murine macrophages. Current Protocols in Immunology (2008).
    2. Boes, M., Prodeus, A.P., Schmidt, T., Carroll, M.C. and Chen, J. A critical role of natural immunoglobulin M in immediate defense against systemic bacterial infection. J. Exp. Med. 188, 2381-2386 (1998).
    3. 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).
    4. 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).
    5. 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).
    6. 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).
    7. 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).
    8. 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).
    9. Stanley, E.R. The macrophage colony-stimulating factor, CSF-1. Methods Enzymol. 116, 564-587 (1985).
    10. 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).
    11. 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).
    12. 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).

    Comments

    52 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
    Reply

    Posted by: AnonymousMarch 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.
    Reply

    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
    Reply

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

    Beautiful tutorial! Thank you!
    Reply

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

    Thanks
    Reply

    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?
    Reply

    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.
    Reply

    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
    Reply

    Posted by: AnonymousJune 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
    Reply

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

    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 propŒr and nice suggestion??
    I will be realy grateful to you.
    Thank you so much.

    Salman
    Reply

    Posted by: Salman K.September 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
    Reply

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

    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 propŒr and nice suggestion??
    I will be realy grateful to you.
    Thank you so much.

    Salman
    Reply

    Posted by: Salman K.September 14, 2010, 4:02 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 propŒr and nice suggestion??
    I will be realy grateful to you.
    Thank you so much.

    Salman
    Reply

    Posted by: Salman K.September 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
    Reply

    Posted by: AnonymousSeptember 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.
    Reply

    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
    Reply

    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
    Reply

    Posted by: AnonymousNovember 2, 2010, 2:38 PM

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

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

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

    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.
    Reply

    Posted by: AnonymousApril 19, 2011, 10:31 PM

    Dear Jamal,

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

    Thanks,

    Avijit
    Reply

    Posted by: AnonymousJune 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
    Reply

    Posted by: AnonymousJune 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
    Reply

    Posted by: AnonymousJune 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 dŒs the mean "macrophages obtained by this procedure differ in some of their physiological properties."?
    Reply

    Posted by: AnonymousJune 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
    Reply

    Posted by: AnonymousJune 19, 2011, 5:57 PM

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

    Posted by: AnonymousJuly 19, 2011, 5:48 AM

    Hi,

    Starch can be used but I prefer thioglycollate.

    Thanks,

    Avijit
    Reply

    Posted by: AnonymousJuly 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
    Reply

    Posted by: AnonymousSeptember 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
    Reply

    Posted by: AnonymousSeptember 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
    Reply

    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
    Reply

    Posted by: AnonymousFebruary 19, 2012, 1:21 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
    Reply

    Posted by: AnonymousFebruary 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
    Reply

    Posted by: AnonymousFebruary 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!

    Reply

    Posted by: AnonymousMarch 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
    Reply

    Posted by: AnonymousMarch 2, 2012, 5:51 PM

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

    Posted by: AnonymousMarch 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
    Reply

    Posted by: AnonymousMarch 7, 2012, 11:38 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?


    Reply

    Posted by: AnonymousMarch 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
    Reply

    Posted by: AnonymousMarch 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.

    Reply

    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

    Reply

    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
    Reply

    Posted by: Inês 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
    Reply

    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
    Reply

    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!
    Reply

    Posted by: Inês S.April 2, 2013, 5:38 AM

    Dear Dr. Ray (and JoVE colleagues who are reading this comment),

    Thank you very much for providing this protocol. I want to use a similar protocol for Sprague-Dawley rats (I'm new to rats and rat anatomy), but I am having two main issues:

    1) the volume of PBS solution I need to inject - the rats are huge and so are their peritoneal cavities.. Is it okay to inject PBS into a rat multiple times without leakage? I think I will need at least 50mL.

    2) opaque peritoneal membrane due to muscle tissue - After I cut the rat skin off, muscle tissue is attached to peritoneal membrane and it's quite difficult to isolate. I tried to inject PBS with muscle tissue, but it's really difficult to see where my needle is going. Because I did not deal with rats before, I would like to have some guidance from you and JoVE colleagues.

    It would be greatly appreciated if I could get a private message / reply regarding this matter. Thank you very much for reading.

    Sincerely,
    Jeannie
    Reply

    Posted by: Jeannie C.June 27, 2013, 10:43 AM

    Dear Dr. Ray

    Thanks for posting this very useful video. As we are new to immunology, we got some difficulties with isolating peritoneal cavity Neutrophils. The protocol we are using involved in injecting 8% casein solution and then wash the peritoneal cavity, just as described in your video. However, we had some problems with making casein solution, as it won't go into PBS solution. I therefore wondered whether you could give us some suggestions, or if you could forward us with your protocol for neutrophil isolation. We sincerely appreciate your help.

    Thanks a lot

    Sincerely

    Chen
    Reply

    Posted by: Chen D.March 27, 2014, 3:25 AM

    Dear Chen,

    Casein is not easily soluble in PBS. To make casein-PBS solution, heat PBS at 80-90 degree C and slowly add small aliquots of casein while stirring. Let the first aliquot go into solution before adding the next one.

    Thanks,

    Avijit
    Reply

    Posted by: AnonymousMarch 28, 2014, 11:29 AM

    Dear Dr. Ray
    Thank you for providing this video. I am a first year PhD student and I have some queries:
    Can you tell me which is the predominant type of macrophage found in peritoneum after giving a thioglycolate injection: M1 or M2 or naive? Are dendritic cells also present?
    Can I co culture these isolated macrophage subtype with some other cells? In this condition, what type of media should I use ?

    Yours sincerely,
    Akanksha
    Reply

    Posted by: akanksha s.June 27, 2014, 2:53 AM

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