Hi guys,
I'm working on the tumor mouse model. In general, I prepared tumor cells and injected cells into the flank of mice subcutaneously.
After around 3 weeks, I found, in the same group, there's a variation in the tumor weight. For instance, some tumor were around 1.2g, some 0.6g.
Could you give me some suggections or tips about how do tumor inejction?
Which type of needle do you need for injection? or How to prepare tumor cells suspension? or whatever something you think is important to do that successfully
Many thanks :)
Dear Tingting Wang
There could be experimental errors like imprecise inoculation of cells or leakage that might lead to variations in tumor size or even the absence of a tumor.
I have listed down some measures which you could incorporate in your protocol.
1. You should inject the same number of cells to each mouse. To do so, you should trypsinize the cells carefully without affecting viability of the cells. The non-viable cells should be disregarded during cell counting. Formation of clumps of cells is another factor that must be considered. You should avoid these clumps by pipetting the cells up and down as formation of cell clumps will cause problems during cell counting and will lead to miscalculation of the cell number.
2. Before adding PBS or media to the pelleted cells that are to be injected, it is important to know the volume of the cells. This may be estimated by comparing the cell pellet with tubes filled with different volumes of media. Subsequently, the cell volume may be subtracted from the intended media volume, thereby adding lesser volume of PBS/media to the cell pellet. You may use the formula provided below.
Volume of media that needs to be added to the cell pellet = Calculated volume - Cell volume.
So, you will avoid adding extra PBS/media to the pellet.
3. The cell pellet may be resuspended in media, PBS or Matrigel for injection. Among these, use of Matrigel is a convenient option as it may minimize cell leakage. In addition, engraftment in the presence of Matrigel may enhance tumor growth and metastatic potential of cells. You may prepare appropriate number of cells into single cell suspension in 50-100μL volume (PBS:matrigel=1:1) and inject subcutaneously on the flank or the abdominal area of the mice.
Please note: Some Matrigel preparations contain growth factors that potentially influence the experimental results. However, growth factor-depleted Matrigel are also available commercially. You may select the type of Matrigel that suits your purpose.
4. Use a 1 ml syringe, and a 25- or 27-gauge needle, draw up the required dose and expel any air bubbles. The injections are usually made under the skin of the back and sides, or under the skin overlying the neck.
5. In most tumor mouse models, 10^5 – 10^6 cells from an established tumor cell line are injected subcutaneously. The volume of cell suspension injected is in the range of 50-100μL and not more than 150μL.
6. You may administer the injection in either one of the two ways.
a. SCRUFF: Inject into the loose skin over the neck. Using your dominant hand, direct the needle into the base of the skin tent to avoid needle stick injury.
b. FLANK: Holding the needle at a very shallow angle, direct it under the skin overlying the upper thigh and lower part of back & abdomen.
You may want to refer to the link below for the diagrammatic representation of administration of injection to mice subcutaneously.
https://www.urmc.rochester.edu/MediaLibraries/URMCMedia/animal-resource/forms/documents/Mouse-SQ-injection-handout.pdf
Some more useful information may be obtained from the link below.
https://staff.flinders.edu.au/content/dam/staff/research/ebi/animal/sops/swms-mouse-injection-techniques.pdf
These are some suggestions that will help you.
Good Luck!
Regards,
Malcolm Nobre
Dear Tingting Wang ,
Good day to you. I just want to share my experience in doing the procedure to you as below,
- use sterile items (forceps, sterile paper, cotton. etc) during the procedure. - sterility is important to avoid unknown mass (pus, infection)
- use isoflurane to sedate the mice.
- disinfect the area (right lower flank) before inoculation. Use 70% alcohol/iodine to disinfect the area
- use forceps to hold the skin to create space of the subcutaneous/loose the skin.
- insert needle, push the cells inside and hold for 5 sec before pull out the needle to avoid leakage. matrix gel help retain the injected cells at the site of inoculation. Without a matrix gel, injected cells might disperse or migrate away from the injection site. (note: some cells are large- can see the sediment at the syringe, just mix and adjust the bubble).
- Place the mice in a sterile cage , monitor by measuring the tumor growth by using caliper perpendicular. Measure in mm3 (w2 x l/2).
during the involvement in study related to the tumor, we experience different morphology of tumor depend on cells we used. Some cells will cause tumor to necrotic, solid, double lobe. Volume/viability cells also important cause it is related to duration of cell growth and morphology of the tumor. The syringes we received marked with number and we will start with number 1 which means it was prepared earlier.
I hope you will get some input from my experience for your study.
Good luck!
Regards,
Nurul
Hi Nurul Nurul Ashikin Mohamed Shahrehan ,
Thank you so much for your suggestions! I didn't think about the position of syringe before, and I think your suggestion would help me a lot!
Do you think that different gauge needles could make a difference? Why do you choose 26G?
We don't have matrix gel, so the cells were resuspended in the PBS.
When I took out the tumor, I found there were more like fat tissue in the tumor, and in some tumors, there's a lot of blood, eventhough I injected the same cells.
Best wishes,
Tingting
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