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 JoVE Immunology and Infection

Isolation of Mouse Peritoneal Cavity Cells

1, 1

1BloodCenter of Wisconsin, Blood Research Institute

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    Summary

    The peritoneal cavity in mammals contains different immune cell populations crucial for innate immune responses. An efficient isolation method is required for biochemical and functional analyses of these cells. Here we provide a comprehensive method for the isolation of peritoneal cavity cells in the mouse.

    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

    The peritoneal cavity is a membrane-bound and fluid-filled abdominal cavity of mammals, which contains the liver, spleen, most of the gastro-intestinal tract and other viscera. It harbors a number of immune cells including macrophages, B cells and T cells. The presence of a high number of naïve macrophages in the peritoneal cavity makes it a preferred site for the collection of naïve tissue resident macrophages (1). The peritoneal cavity is also important to the study of B cells because of the presence of a unique peritoneal cavity-resident B cell subset known as B1 cells in addition to conventional B2 cells. B1 cells are subdivided into B1a and B1b cells, which can be distinguished by the surface expression of CD11b and CD5. B1 cells are an important source of natural IgM providing early protection from a variety of pathogens (2-4). These cells are autoreactive in nature (5), but how they are controlled to prevent autoimmunity is still not understood completely. On the contrary, CD5+ B1a cells possess some regulatory properties by virtue of their IL-10 producing capacity (6). Therefore, peritoneal cavity B1 cells are an interesting cell population to study because of their diverse function and many unaddressed questions associated with their development and regulation. The isolation of peritoneal cavity resident immune cells is tricky because of the lack of a defined structure inside the peritoneal cavity. Our protocol will describe a procedure for obtaining viable immune cells from the peritoneal cavity of mice, which then can be used for phenotypic analysis by flow cytometry and for different biochemical and immunological assays.

    Protocol

    1. Before starting the process, the following items need to be to collected and prepared:
      • Ice
      • 5ml syringe with 27g needle
      • 5ml syringe with 25g needle
      • Styrofoam block and pins for mounting the mouse
      • Tray on which the mounting block can be placed
      • Scissors and forceps
      • Collection tubes
      • 70% ethanol
      • PBS with 3% fetal calf serum (FCS) (Pre chilled and kept on ice)
    2. Euthanize the mouse, spray it with 70% ethanol and mount it on the styrofoam block on its back.
    3. Using a scissors and forceps cut the outer skin of the peritoneum and gently pull it back to expose the inner skin lining the peritoneal cavity.
    4. Inject 5 ml of ice cold PBS (with 3% FCS) into the peritoneal cavity using a 27g needle. Push the needle slowly in the peritoneum being careful not to puncture any organs.
    5. After injection, gently massage the peritoneum to dislodge any attached cells into the PBS solution.
    6. Insert a 25 g needle, bevel up, attached to a 5 ml syringe in the peritoneum and collect the fluid while moving the tip of the needle gently to avoid clogging by the fat tissue or other organs. Collect as much fluid as possible and deposit the collected cell suspension in tubes kept on ice after removing the needle from the syringe.
      Optional: Repeat step 4-6
    7. Make an incision in the inner skin of the peritoneum and while holding up the skin with a forceps use a plastic Pasteur pipette to collect the remaining fluid from the cavity.
    8. If in step 6 or 7 visible blood contamination is detected then the contaminated sample should be discarded.
    9. Spin the collected cell suspension at 1500 RPM for 8 minutes, discard the supernatant and resuspend the cells in desired media or PBS for counting.

    Alternative protocol to obtain thioglycollate elicited macrophages:

    This method is used to obtain a higher yield of macrophages.

    1. Inject 5 ml of 3% (w/v) Brewer thioglycollate medium (7) into the peritoneal cavity of each mouse.
    2. Wait for 3-5 days, and proceed to step 1 above for the collection of the cells.

    Compared to the nonelicited approach, approximately 10 times more macrophages can be collected. The only concern is that macrophages obtained by this procedure differ in some of their physiological properties.

    Representative results:

    From an unmanipulated mouse, 5-10 million peritoneal cavity cells can be obtained with a good isolation protocol. Among all live cells, 50-60% are B cells, ~30% macrophages and 5-10% are T cells (Fig 1).

    Figure 1
    Figure 1. Phenotype of cells isolated from the peritoneal cavity. Following isolation of peritoneal cavity cells they were stained with anti-mouse TCRβ-FITC, B220-PE-Texas red, CD11b-Pacific blue, CD23-PE-Cy7 and CD5-APC. Representative flow cytometric plots show percentages of B and T cells (a), macrophages (b), B1 and B2 cells (c) and B1a and B1b cells (d).

    Discussion

    Isolation of peritoneal cavity cells is an important technique for the study of different immune cells, primarily macrophages and specific B cell subsets. Although this is a simple process, there are some critical steps involved. Presence of contaminating blood in the collected sample should be avoided to obtain a pure peritoneal cavity cell population. Euthanasia by cervical dislocation should be done carefully to avoid blood contamination in the peritoneal cavity. Alternatively CO2 can be used. In addition, during the procedure proper care should be taken to avoid puncturing the bladder or any other organs in the peritoneal cavity. Recovering most of the injected fluid is also important to the cell yield.

    This procedure is widely used to study macrophage biology since moderate numbers of resident, unstimulated macrophages can easily be obtained from the peritoneal cavity, in contrast to the laborious task of differentiating myeloid progenitor cells into mature macrophages in vitro, using macrophage colony stimulating factor (8,9). The macrophage yield from the peritoneal cavity can be improved by using the thioglycollate elicitation method, although thioglycollate use might alter physiological properties of the macrophages (7).

    B cells are an important part of our immune system playing crucial roles in both innate and adaptive immunity. Among different B cell subpopulations, B1 cells comprise a unique subset of B cells involved in innate immunity, autoimmunity and immune regulation. B1 cells are primarily located in the peritoneal cavity and are self replenishing. They are one of the primary producers of natural IgM which provides the first line of protection against a number of virus and bacteria (10-12). B1 cells are also a major source of IL-10 (6), which is an important cytokine involved in immune regulation. Although several studies have been pursued with the B1 cells, still there is scope for further evaluation of their contrasting functions, specially their regulatory role. Peritoneal cavity cell isolation method provides the unique opportunity to study the functions of B1 cells.

    Disclosures

    Acknowledgements

    This work was supported in part by NIH grant AI069358 and the BloodCenter Research Foundation.

    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

    Thanks so much!
    Reply

    Posted by: AnonymousJune 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.
    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 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
    Reply

    Posted by: AnonymousJanuary 3, 2011, 9:21 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, 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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