Navigate this Article


Subcutaneous Injection of Tumor Cells   

How to cite Favorites 2 Q&A Share your feedback Cited by


Growth of cells in the subcutaneous space of immunocompromised mice is a common method for assaying tumorigenic potential in vivo. This technique is also used to assess the effects of therapeutic interventions on cancer cell lines.

Materials and Reagents

  1. Tumor cells
  2. CB17 scid/scid mice
  3. Trypsin (Life Technologies, InvitrogenTM, catalog number: 25300-054 )
  4. DMEM (Life Technologies, InvitrogenTM, catalog number: 11965-092 )
  5. Fetal bovine serum (FBS) (Life Technologies, InvitrogenTM, catalog number: 16000-044 )
  6. Phosphate buffered saline (PBS)
  7. Trypan blue (Life Technologies, InvitrogenTM, catalog number: 15250-061 )
  8. Isoflurane (usually purchased through animal facility at institution)
  9. Matrigel (BD Biosciences, catalog number: 356234 )
  10. DMEM cultural medium (see Recipes)


  1. Centrifuges
  2. Insulin syringe
  3. Hemocytometer
  4. Cell culture hood
  5. Incubator
  6. Microscope
  7. Small gauge
  8. Eppendorf tube


  1. Remove growth medium from cells and wash with 5 ml of PBS.
  2. Aspirate PBS, add 2 ml of trypsin and incubate for 5 min, or until cells have detached, at 37 °C.
  3. Quench trypsin by adding at least 3 volumes of 10% FBS containing medium.
  4. Pellet cells by centrifugation for 5 min at 1,100 rpm and 37 °C.
  5. Aspirate medium, wash cells with 10 ml sterile PBS, mix well with pipette and save 50 μl aliquot of cells for counting.
  6. Pellet cells by centrifugation for 5 min at 1,100 rpm and 37 °C.
  7. While cells are spinning, add 50 μl of trypan blue to saved aliquot, mix well and count cells using a hemocytometer.
    Note: Dark blue cells are dead and should not be counted.
  8. Aspirate PBS, resuspend cells in fresh PBS to a concentration 1 x 106 cells/100 μl and transfer cells to a sterile Eppendorf tube.
    Note: The cell number required depends upon the aggressiveness of the tumor cells and can vary by an order of magnitude.
  9. (Optional) Add an equal volume of thawed Matrigel to cells and mix carefully with pipette. Important, Matrigel should be thawed on ice because it will solidify at room temperature.
    Note: Matrigel provides a favorable environment for less aggressive cells to grow and is often used.
  10. Slowly pull up 100 μl of cells alone or 200 μl of cell/matrigel mixture using an insulin syringe.
    Note: Cells can be damaged by the small gauge of the insulin needle; however, insulin syringes provide a more accurate volume measurement. If significant death is observed, 1 ml syringes with 22 gauge needles can be substituted. Place cell containing syringes on ice to prevent matrigel from polymerizing.
  11. Inject 1 x 106 cells into the flanks of immune deficient mice, preferably CB17 scid/scid. To do this, pinch the skin of the mouse between your index finger and thumb and pull the skin away from the body of the mouse.
  12. Inject slowly and evenly into the pouch created by your fingers, creating a single bubble of cells beneath the skin and avoiding too much spread of the cells.
  13. Anesthetizing the mice using isoflurane makes the injection process significantly less stressful for the both the mice and the researcher.


  1. DMEM cultural medium
    Supplemented with 10% FBS
Please login or register for free to view full text
Copyright: © 2011 The Authors; exclusive licensee Bio-protocol LLC.
How to cite: Reuter, J. (2011). Subcutaneous Injection of Tumor Cells. Bio-101: e166. DOI: 10.21769/BioProtoc.166.

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 including images for the troubleshooting.

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.

ngu trinh
trinh van ngu
why need to use Matrigel in this experiment? what is the function of Matrigel? Any research did without Matrigel?
12/10/2018 5:43:40 PM Reply
shohreh fakhari
kurdistan university of medical sciences

Matrigel has been shown to significantly enhance the tumorigenicity of a wide range of cancer cell lines in vivo.
if you don't want to use matrigel, you should use large number of cells for increased tumorigenicity of cancer cells.

12/11/2018 3:00:33 AM

ngu trinh
trinh van ngu

Thank you so much for your response.

12/14/2018 6:17:25 PM

What can happen if you inject the tumor cells in complete medium? Will this affect the experiment?
Thank you!
7/10/2014 11:36:48 PM Reply
Jason Reuter

You should be consistent between, but I expect the experiment will work. It should also be acknowledged that tumor growth may depend on serum and medium.

7/10/2014 11:49:13 PM


In which way may the tumor growth depend on the serum? Will not it be drain out if it is in small volum(100ul)? Thx

7/10/2014 11:59:23 PM

Jason Reuter

The fluid will be absorbed. However, it may support the cells initially. My only point is that you haven't strictly tested the cells' ability to grow tumors independent of additional supplements.

7/11/2014 12:28:01 AM


ok. Thank you very much for your advice and quick reply.

7/11/2014 12:35:30 AM

We use cookies on this site to enhance your user experience. By using our website, you are agreeing to allow the storage of cookies on your computer.