Xenografting protocol

Xenografting protocol

Postby cell2bio on Wed Jul 09, 2014 4:18 pm

Hi all - looking for some advice on a preliminary study of tumor growth. I am doing a study with ten NOD/SCID mice. All mice have a 0.72 mg pellet of estrogen inserted between their shoulder blades, and after two days they received a xenotransplant of 5 x 10^6 MCF-7 cells with an inducible plasmid.

An aside about the cells - they were cultured normally, treated with trypsin, centrifuged down, washed and resuspended in sterile phosphate-buffered saline. They were counted using a hemocytometer, and the concentration was adjusted to that they were the correct concentration in 200 microliters of PBS. This solution was then inserted subcutaneously into the left flank of the mice I described above.

The issue is that a month later, there's no tumor growth observed. This is the second time I've attempted this protocol, but the first was with RAG-knockout mice, who also did not develop tumors (although they did get an inflamed uterus).

Did I do something wrong?
cell2bio
 
Posts: 7
Joined: Tue Jun 24, 2014 2:29 pm

Re: Xenografting protocol

Postby medicbio on Wed Jul 09, 2014 5:13 pm

Here's my typical protocol:
1) Decide the number of cells for injection (ie 5x10^6 ) to calculate the number of plates that will require trypsinizing.
(typically a 100% confluent plate of 100 mm^2 yields at least 2 grafts at 5x10^6 cells/injection)
2) Trypsinize the number of plates you calculated in step 1.
3) Collect trypsinized, loose cells in 50 ml tube and centrifuge for 4 minutes at 800 RPM.
4) Remove supernatant and resuspend in 25 ml of SFM to count cells.
5) Remove three 100 microliter aliquots into 3 separate tubess and dilute each 100 microliter 1:5 by adding 400 microliter of SFM and mixing well.
6) Remove 50 microliter of each 1:5 dilutions for counting, count each and then get the mean of the three numbers.
7) Determine the concentration of cells in cells/ml by using the following formula:
Average counts x 10,000 x dilution factor of 5 = number of cells/ml.
8) Calculate the volume required to add to achieve final concentration of cells for injection per chosen volume to be injected (ie 5 x 10^6 cells/ 100 microliter injection) by initially determining the total number of cells in the 25 ml suspension. Calculate this by multiplying the concentration of cells in #cells/ml x 25 = total number of cells.
Then use the following equation:
Total # cells/x volume = 5x10^6 / 100ul
Solving for x gives you the volume to resuspend pellet of cells to achieve desired final concentration (in this example 5x10^6 cells/ 100 microliter).
9) Centrifuge down the 50 ml tube for 4 minutes at 800 RPM.
10) Discard supernatant and resuspend the pellet in the previously determined volume from step #8.
11) Draw up each injection you need a 1 ml syringe in the sterile hood prior to going to the animal facility. Place the separate syringes each containing 100 microliters (or your desired volume) on ice to minimize cells settling after resuspension which would alter the concentration of cells.
12) Anesthetize each mouse with inhaled isoflorane just prior to injection. Be careful with the isoflurane as the mice may succumb to respiratory depression. Cease administration when they just begin to stop moving and remove them from the source of anesthetic, let them breathe pure air for a few seconds then place their noses just adjacent to the opening of the isoflurane tube during the injection.
13) Inject into desired area.

This looks similar to yours and always works. Perhaps something is off with your cell line?
medicbio
 
Posts: 7
Joined: Tue Jun 24, 2014 2:23 pm


Return to Cancer and Tumor Biology

Who is online

Users browsing this forum: No registered users and 1 guest

cron