Welcome guest, Login | Register

Home

X
加载中

Peritoneal macrophages are used as primary macrophages in lots of studies, mainly because they are easy to obtain. Injection of thioglycollate broth i.p. induces inflammatory responses and elicits large numbers of macrophages. This protocol can be used for harvesting resident or thioglycollate-elicited peritoneal cells. Peritoneal macrophages are non-adherent in situ and when they are cultured in dishes, they become adherent so that macrophages may be separated from other types of cells in peritoneal cavity.

Thanks for your further question/comment. It has been sent to the author(s) of this protocol. You will receive a notification once your question/comment is addressed again by the author(s).
Meanwhile, it would be great if you could help us to spread the word about Bio-protocol.

X

Harvest and Culture of Mouse Peritoneal Macrophages

Cell Biology > Cell isolation and culture > Cell isolation
Authors: Mingfang Lu
Mingfang LuAffiliation: Internal Medicine, University of Texas Southwestern Medical School, Dallas, TX, USA
Bio-protocol author page: a990
 and Alan W. Varley
Alan W. VarleyAffiliation: Internal Medicine, University of Texas Southwestern Medical School, Dallas, TX, USA
For correspondence: alanwaynevarley@gmail.com
Bio-protocol author page: a991
Vol 3, Iss 22, 11/20/2013, 10346 views, 1 Q&A, How to cite
DOI: http://dx.doi.org/10.21769/BioProtoc.976

[Abstract] Peritoneal macrophages are used as primary macrophages in lots of studies, mainly because they are easy to obtain. Injection of thioglycollate broth i.p. induces inflammatory responses and elicits large numbers of macrophages. This protocol can be used for harvesting resident or thioglycollate-elicited peritoneal cells. Peritoneal macrophages are non-adherent in situ and when they are cultured in dishes, they become adherent so that macrophages may be separated from other types of cells in peritoneal cavity.

Materials and Reagents

  1. C57BL/6 mouse
  2. 70% alcohol or isopropanol
  3. PBS (Life Technologies, catalog number: 10010023)
  4. EDTA (Life Technologies, catalog number: 15575-020)
  5. RPMI 1640 (Cellgro®, catalog number: 10-041-CV)
  6. 10% heat-inactivated FBS (endotoxin < 0.06 EU/ml) (Hyclone, catalog number: SH30071.03HI)
  7. Penicillin-Streptomycin-Glutamine (Life Technologies, catalog number: 10378016)
  8. Nonessential amino acids (Life Technologies, catalog number: 11140050)
  9. Sodium pyruvate (Sigma-Aldrich, catalog number: S8636)
  10. Hepes (suitable for cell culture) (Sigma-Aldrich, catalog number: H0887)
  11. 2-mercaptoethanol (EMD Millipore, catalog number: ES-007-E)
  12. Thioglycollate medium (BD Biosciences, catalog number: 211716)
  13. Pyrogen-free water (Hyclone, catalog number: SH30529.03)
  14. cRPMI medium (see Recipes)

Equipment

  1. 5 ml syringe
  2. 12-well plate
  3. 15 ml sterile Falcon tubes
  4. Needles (20 G and 25 G)
  5. Centrifuge
  6. 37 °C, 5% CO2 cell culture incubator
  7. 300 ml flask with an aluminum foil lid
  8. Laboratory oven

Procedure

  1. Harvest and culture of resident peritoneal cells
    1. Fill 5 ml of PBS with 5 mM EDTA in a 5 ml syringe with a 25 G needle.
    2. Anesthetize and sacrifice mouse with CO2.
    3. Place mouse with abdomen up on paper towel in hood.
    4. Swab abdomen with 70% alcohol or isopropanol.
    5. Make a small incision in the center of the skin overlying the peritoneal wall.
      Note: Small incision is made on the skin to peel the skin off. Injection and extraction can be at different sites on peritoneal membrane.
    6. Firmly pull skin to expose the peritoneal wall.
    7. Insert the needle to peritoneal membrane; avoid inserting needle into guts or bladder. Inject 5 ml of PBS EDTA into peritoneal cavity.
    8. Massage abdomen for approximately 10-15 sec.
    9. Withdraw the needle slowly. Change the 25 G needle on the syringe to a 20 G needle.
    10. Use one hand to push the fluid to one side of the peritoneum. Using the other hand, insert needle to the side of cavity with plenty of fluid and withdraw the fluid from peritoneum. Avoid fat, gut or mesentery, which may clog the needle. Try to draw as much fluid as possible. Usually, approximately 4 - 4.5 ml fluid can be recovered from one mouse.
    11. Remove needle from syringe and dispense contents into a centrifuge tube on ice.
    12. Centrifuge peritoneal cells (300 x g; 3 min) and collect cell pellet. Usually about 2-4 million resident peritoneal cells can be recovered from one C57BL/6 mouse using this method and about 50% are peritoneal macrophages.
    13. Resuspend cell pellet from one mouse in 1 ml of cRPMI medium, count cells.
    14. Culture peritoneal cells in a 12-well plate, 2 million/well in 1 ml cRPMI at 37 °C with 5% CO2 for 6-18 h. During this time, peritoneal macrophages adhere to the plastic surface. The floating non-macrophages can then be washed away by adding and aspirating 0.5 ml cRPMI medium twice.
    15. The adherent macrophages are ready to use.

  2. Harvest and culture thioglycollate-elicited peritoneal cells
    Thioglycollate elicited peritoneal macrophages can be harvested and cultured in the same way as described in steps 1-15 with additional steps as shown below.
    1. Preparation of 3% thioglycollate medium.
      1. Heat a 300 ml flask with an aluminum foil lid (180-200 °C) in a laboratory oven for at least 18 h to get rid of endotoxin.
      2. Suspend 6 grams of thioglycollate medium in 200 ml of pyrogen-free water.
      3. Autoclave (15 psi/121 °C/15 min).
      4. After cooling, aliquot to 15 ml sterile Falcon tubes. Store in a dark place at room temperature for 2 months before using. We have found that thioglycollate medium stored at room temperature for up to 2 years can still be used.
    2. Inject 1 ml of aged thioglycollate i.p. per mouse. Wait for 4-5 days, harvest peritoneal cells. About 10 million macrophages can be recovered from one mouse.

    Note: The study was performed under an IACUC approved protocol (LCID 11E).

Recipes

  1. cRPMI medium
    RPMI 1640 with 10% heat-inactivated FBS (endotoxin < 0.06 EU/ml)
    2 mM L-glutamine
    100 μM of nonessential amino acids
    100 U/ml penicillin
    0.1 mg/ml streptomycin
    10 μM of sodium pyruvate
    25 mM Hepes, pH 7.4
    50 μM 2-mercaptoethanol

Acknowledgments

This research was supported by the Intramural Research Program of the NIH, NIAID

References

  1. Lu, M., Varley, A. W. and Munford, R. S. (2013). Persistently active microbial molecules prolong innate immune tolerance in vivo. PLoS Pathog 9(5): e1003339.


How to cite this protocol: Lu, M. and Varley, A. W. (2013). Harvest and Culture of Mouse Peritoneal Macrophages. Bio-protocol 3(22): e976. DOI: 10.21769/BioProtoc.976; Full Text



Reproducibility Feedback:

  • Add Photo
  • Add Video

Bio-protocol's major goal is to make reproducing an experiment an easier task. If you have used this protocol, it would be great if you could share your experience by leaving some comments, uploading images or even sharing some videos. Please login to post your feedback.

Q&A and Troubleshooting:

  • Add Photo
  • Add Video

Please login to post your questions/comments. Your questions will be directed to the authors of the protocol. The authors will be requested to answer your questions at their earliest convenience. Once your questions are answered, you will be informed using the email address that you register with bio-protocol.
You are highly recommended to post your data (images or even videos) for the troubleshooting. For uploading videos, you may need a Google account because Bio-protocol uses YouTube to host videos.


Login | Register
7/13/2016 8:03:47 PM  

wang liang
zhejiang hospital

Hi,Thank you for posting this protocol.
I gotta a question about Procedure B. After procedure B,what types of macrophage can we get ? M0,M1 or M2?
Thank you for answering my question,

Liang

The latest modification time: 7/13/2016 8:15:31 PM

Reply

Please login to post your questions/comments. Your questions will be directed to the authors of the protocol. The authors will be requested to answer your questions at their earliest convenience. Once your questions are answered, you will be informed using the email address that you register with bio-protocol.
You are highly recommended to post your data (images or even videos) for the troubleshooting. For uploading videos, you may need a Google account because Bio-protocol uses YouTube to host videos.

Login | Register

Share
Twitter Twitter
LinkedIn LinkedIn
Google+ Google+
Facebook Facebook