I'm growing 4T1 cells in mice. I'm implanting two contralateral tumors per mouse. I'm having a hard time getting the tumors to grow symmetrically. I understand they won't all be perfect, but I'm getting a lot of shapes that are difficult to caliper, or I'll have tumors with small satellite tumors right next to them. With 2 tumors on 1 mouse, its hard to get enough mice to enroll in study.
I'm implanting 1 x 10^5 cells in 100 uL volumes. I draw the cells up into the syringe in an 18 g needle, and I implant the cells using a 25 g needle. I slowly inject the tumors on the flank and slowly remove the syringe. The cells are being implanted using serum free media.
Should I use a smaller gauge (27g) needle to implant? Should I move my implant site up to the shoulder? Should I switch to PBS instead of media?
Hi,
You can inject 4T1 cells from 5x103 to 1x105 in the up or low fat pad with volume 30ul to 50ul in Balb/c mice. Then you can have lung metastasis and larger spleen ( 3-4 time weight (g) than normal mouse''s spleen.
Hello Derek,
4T1 forms tumors of multiple sahpes and sizes. In my experience tumors were mostly oval or nearly spherical in shape. However i was using a cell number 10 time higher than what you are using. Twin tumors mostly occur when during injection cells are unloaded at two adjecent sites. We inject cells in the Lowe adominal mammary fat pads and most animals (approx 70%) develop visible tumors within 7 to 10 days. There are few sugestions-
1. Use antibiotic free medium for preparing cell suspension. Prefer white media or else PBS.
2. Always use cells in growth phase, grow the cells to 60%confluency and 6 hour prior to injection add fresh meda.
3. Make sure you are injecting at one site only. You can use suitable animal restrainers if requiered.
Best
Hello
I dont have experience in this field but I read in an article by ( Michael K , et al ) used a forceps, gently grasp and lift the 4th inguinal mammary pad. Carefully, place a 27.5 gauge needle bevel side up and inject into the mammary fat pad directly under the nipple, taking special care not push the needle into the abdominal cavity. after that release the gland and slowly inject 50 μl of the cell suspension (1x104 cells) into the mammary fat pad.
NOT :
in attachments a number of protocols I hope help you
Good luck in your experience
Orthotopic transplantation is the most reliable method to perform. 1-5 X10 (5) cells are enough for each mammary gland to form the tumor tissue and invasive loci in the lungs.
http://www.nature.com/ncomms/journal/v3/n6/fig_tab/ncomms1892_F2.html
Hi Derek,
We commonly do 4T1 tumors on bilateral flanks. I only inject about 500,000 cells and sometimes even half of that as they grow so vigorously. 500,000 cells will give us tumors easily in one week (100%). 250,000 cells will result in small tumors in about 7 to 10 days. Cells are suspended in PBS and then drawn up in a syringe without a needle. Then I attach a 27 ga needle to do the injections - 100ul per tumor. I think one of the problems may occur if you are sticking the needle too far under the skin. The cells seem to follow the path of the needle stick so only inject a short distance to keep the cells sequestered in a small location. The other precaution is to keep the cells resuspended as they seem to settle out rather quickly. I pull them up and down in the syringe without needle to resuspend the cells between injections.
Hi
I worked with 4T1 cells but I injected orthtopically, into the mammary fat pad of the mouse and they grew well. We also studied this cell line for metastasis in lung and liver tissues.
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