In this video, we demonstrate a protocol to establish mouse thymic lymphoma. Cell lines. Tumors were harvested for mice age three to six months at the earliest syndication of visible tumors.
Based on the observation of hunched posture, labored breathing, poor grooming, and wasting, we have successfully established several ATM null and P 53 null thymic lymphoma cell lines Using this protocol. In part one, we describe how to harvest the thymic lymphoma from a tumor bearing.Mouse. Mice for this protocol are routinely euthanized with carbon dioxide.
Place a clean paper towel on the dissection pad and spray with 70%ethanol. Place the euthanized mouse on the dissection pad. Spray both sides of the animal with ethanol.
Pin the mouse onto the dissection pad and spray thoroughly with 70%ethanol. Open the skin using sterile scissors and forceps while taking care not to penetrate into body cavities. Open the abdominal cavity with a new pair of sterile instruments.
Take care not to damage any organs. Open the thoracic cavity by dissecting and deflecting the rib cage to reveal the tumor without damaging any blood vessels. This mouse has a well-developed tumor occupying the entire thoracic cavity.
Separate the tumor from other organs and excise a piece approximately 40 to 50%of the volume of the tumor. Transfer the Piece of tumor into cold PBS. Take another piece of the tumor and fix it in 10%formin.
For histology, you may save the remaining portion of the tumor. For other assays, you additionally may examine and fix other organs at this time. In this section, we describe how to culture the thymic lymphoma cells from harvested tumors.
Braid the tube containing the dissected tumor with 70%ethanol and transfer it into a tissue culture hood. Add 10 milliliters of culture medium to the sterile Petri dish. Transfer the piece of tumor into Petri dish with gentle suction from the pipette bend two sterile 18 gauge needles facing the Beveled side out.
Dissociate the piece of tumor using the bent needles. Swirl the dish tap gently and place at an angle to collect the cell suspension to a side while avoiding large tumor pieces. Aspirate the cell suspension and transfer into a 50 milliliter conical tube.
While avoiding large tumor pieces. Wash the plate with 10 milliliters of medium and transfer into the same tube. Avoid large tissue Particles.
Mix the cell suspension gently and transfer five milliliters into a T 25 tissue culture flask. Transfer 10 milliliters into a T 75 tissue culture flask. Use the remaining self suspension to make 2 4 10 101, 000 Fold dilution Plate two milliliters of each dilution into individual wells of a six Well plate Gently swirl the plate, incubate the cells for 48 hours.
In this section, we demonstrate how to feed the cultures until they're ready to passage. After 48 hours of incubation, add five milliliters of medium into the T 25 flask. Add 10 milliliters of medium into the T 75 flask along the wall with minimal disturbance to the cells.
Add two milliliters of medium to each well. Of the six well plate along the wall incubate the cells for 72 hours. In this section, we will describe the procedures for packaging and maintaining the established cell lines.
At the end of five days, passage the cells at a one-to-one ratio and retain the original flask For the T 25 flask. Transfer five milliliters of cell suspension into a new flask containing five milliliters of Medium. Add five milliliters of medium into the original flask For the T 75 flask.
Transfer 10 milliliters of cell suspension into a new flask containing 10 milliliters of medium. Add 10 milliliters of medium into the original flask. For the six well plate transfer two milliliters of cell suspension from each well into the corresponding well of a new plate containing two milliliters of medium In each well.
Add Two milliliters of medium into each well of the original plate. Incubate the cells for 72 hours. In our experience, most cell lines were established from these passage.
One cultures at the end of 72 hours. Observe the flasks and plates for the presence of loosely adherent suspension. Cell aggregates over the attached stromal cells.
This is an early indication of an established cell line for the cultures with fewer loosely adherent suspension. Cell aggregates feed the flasks or plates by replacing 50%media every 72 hours taking care not to remove cells passage. Establish cell lines into new T 25 flask.
The cell lines have a doubling time of 18 to 24 hours. The cultures are routinely maintained at a density of one to 3 million cells per milliliter and passage every three days. Add the required volume of previous passage culture to new medium to make up to 10 milliliters, Feed and retain the original culture.
After a few passages, the non-adherent cells will self sustain without the adherent stromal cells and a smaller number of adherent cells will be carried over with each passage. In our experience, most cell lines became established between passage two and eight. Beyond this point, the cultures can be maintained in non tissue culture treated T 25 flasks.
The established cell lines were routinely characterized for cell surface markers by flow cytometry for most of the cell lines, more than 80%of the population with CD three, CD four, and CD eight positive T cells. In the final section, we describe how to prepare freezer stocks for liquid nitrogen storage and how to recover the cell lines. The cell lines are routinely frozen in culture medium with 20%fetal bovine serum and 10%dimethyl sulfoxide two one milliliter.
Freezing vials are prepared from each 48 hour old T 25 flask. Cells are centrifuge at 1500 RPM for five minutes of room temperature. Supernat is discarded and cells are resuspended in cold freezer, medium, immediately frozen at negative 80 degrees Celsius and transferred to liquid nitrogen.
The following day, cells are recovered from liquid nitrogen storage by quickly thawing a vial Resus suspending the contents in 10 milliliters of prewarm culture, media, and culturing for 72 hours before ing several thymic lymphoma cell lines are in culture over one year after being established under this protocol in our laboratory. We wish you good luck in your experiments.
