Hello Melissa Thone

1. Do you keep the cancer cells warm or on ice prior to inoculation? Why?

You should keep cancer cells on ice to maintain the viability of the cells temporarily. You should inject cells as quickly as possible after preparation because viability slowly decreases over time, even on ice.

2. What gage needle do you use (for subcutaneous)? Why?

You should use a 25g 5/8” needle for subcutaneous application.

For i.p. injections of adult mice, you may use 26 g 1/2” needle, for i.v. injections, you may use 26-27g 1/2” needle and a 30g 1/2” needle for some orthotopic injections.

You need to know that the higher the gauze number, the smaller the diameter of the needle. This will then exert more stress on the material and any cells in it. High shear stress and pressure can decrease cell viability.

The lower the gauze number, the stronger the needle, resulting in less chance of bending or breaking.

3. Do you load the needle any special way to avoid shearing?

Hold a mouse with one hand, use the other hand to slide needle 5 to 10 mm subcutaneously and inject 50-100 µl cell suspension (Tumor cells - 1x10^5 - 1x10^6 cells). You should watch for appearance of a bleb. Failure to insert needle far enough will result in leakage of tumor suspension when mice massage the area after injection.

Some comments that could be helpful:

1. Do not let the syringe with cell suspension sit undisturbed on bench for more than 5 minutes because the cells can settle quickly.

2. Be sure to resuspend cells well in the syringe barrel before each injection.

3. Do not pass the cells through needle more than once since this will shear cells and reduce total cell number.

Also, please refer to the article attached below for more information.

(See basic protocol 1)

Article Mouse Model for Pre-Clinical Study of Human Cancer Immunotherapy

Best.