The overall goal of this procedure is to study the evolutionary conserv ability of the heat shock protein G 96 to promote antigen specific CD eight T cell responses in the frog xenopus sleeves. This is accomplished by first harvesting peritoneal leukocytes by peritoneal lavage. The second step of the procedure is to pulse the peritoneal leukocytes with tumor derived GP 96 that chaperone minor and tumor antigens after incubation and washes.
The peritoneal leukocytes are adoptively transferred by intraperitoneal injection into naive frogs in order to prime these frogs against tumor or minor histocompatibility antigens. The third step of the procedure is to either skin graft or tumor challenge the primed frogs and to monitor the onset of skin graft, rejection, or tumor appearance. The final step of the procedure is to obtain peripheral blood for characterizing the T cells involved in the skin rejection and anti-tumor responses.
Ultimately, results can be obtained that show significant acceleration of skin graft rejection, as well as delays in tumor appearance in GP 96 primed frogs through the skin grafting and the tumor challenge assays respectively. Hi, I'm Christina Koska in the lab of Dr.Jacque Robert in the Department of Microbiology and Immunology at the University of Rochester Medical Center. I am Tanya Cruz also from Dr.River's lab.
Today we will show you a procedure for priming clone frogs by adoptive transfer of peritoneal leukocytes, followed by skin grafting and tumor transplantation assays. In addition, we'll also show you how to get peripheral blood from this animals. We use this procedure in our atory to study the ability of heat your protein GP 96 to promote antigen specific CDAT cells in Salla as well to study the cell types involved.
Okay, so let's get started. The frogs used in our experiments are ISO genetic clones from our breeding colony at the University of Rochester. To elicit peritoneal leukocytes or pls for harvesting, use a 25 gauge five eight needle to inject the anesthetized frog intraperitoneal with a heat killed e coli preparation Three days after injection pls are harvested by intraperitoneal lavage.
To begin this procedure, disinfect the abdomen of the anesthetized frog with a small amount of 70%ethanol. Then use an 18 gauge 1.5 needle to inject five milliliters of sterile amphibian phosphate buffered saline, or a PBS pre-war at room temperature into the peritoneal cavity. Remove the needle and gently massage the frog for a minute to ensure that the injected buffer equates with the fluid in the body cavity.
Next, insert a new 18 gauge 1.5 needle without a syringe into the frog's abdomen. While avoiding the blood vessels, collect the peritoneal fluid that will drip from the back of the needle into a clean 50 milliliter conical tube. Make sure to retrieve as much of the initial injected volume as possible after pls have been harvested.
Put the frog in a container with shallow water until it is awake, at which point it can be placed back in its cage. Once the pls have been harvested, centrifuge at 1000 RPM for 10 minutes at four degrees Celsius, remove the sup, natant and resus. Suspend the pls in cold A PBS.
Transfer the RESUSPENDED pls to a 1.5 milliliter micro refuse tube. To pulse the PLS with GP 96. Add the appropriate amount of GP 96 to the pls and mix by pipetting incubate on ice for one hour after one hour.
Centrifuge the cells at 14, 000 RPM for one minute. To remove any unbound GP 96, remove the supernatant. Add cold A PBS and resus.
Suspend the PLS centrifuge again. Wash the S with cold A-P-B-S-A total of three times to ensure there is no residual G 96 left after the final wash. Resus suspend the pulsed pls at a concentration of five times 10 to the five pls per 300 microliters of a PBS to adoptively transfer pls into naive LG six recipients.
Intraperitoneal inject 300 microliters of pulsed pls per animal. Frogs need to be primed at least three days before the skin graft or tumor challenge experiments, which will be shown next. The skin for grafting is obtained from an anesthetized LG 15 donor frog.
Use scissors to cut and remove a small piece of ventral skin, which is the abdominal skin that appears silvery. Due to the presence of ARI four pigmented cells while handling the graft. It is crucial to have as little manipulation of the tissue as possible since that will cause graft damage.
Hold the skin very gently with the forceps and place it in a Petri dish containing cold A PBS. Keep the Petri dish on. Place the skin on a fresh glass slide and use a razor to cut individual grafts into five millimeter by five millimeter pieces.
Keep the fragments in a PBS on ice to graft the skin onto the LG six recipient frog. First, make a small incision on the dorsal skin of the anesthetized recipient. Then insert the five millimeter by five millimeter graft under the skin with the silvery side up.
Make sure to note the scissor and forcep markings on the grafts because those are not counted as graft rejection. Once the scoring starts 24 hours later, a part of the overlaying host's skin needs to be removed from the graft. Use scissors to cut out a window around the graft so that the graft can be freely visualized.
Be careful not to touch the donor skin or to induce bleeding. Start scoring the graft right away by taking a sketch. Skin graft.
Rejection is determined by the percent of destruction of the aridol on the grafted skin. Graft should be checked every two to three days under the stereo microscope. Prepare tumor cells for transplantation by resus, suspending them in tumor culture Medium at a density of five times 10 to the five cells per 300 microliters.
Transplant the cells by subcutaneous injection on one side of the dorsal side of the animal. Five times 10 to the five cells in 300 microliter volume is injected per frog. Tumor growth will start within two to three weeks after tumor challenge.
Note the initial tumor appearance. Use calipers to measure the dimensions of the tumor and record its volume every two to three days prior to the collection of blood leukocytes from the frog. Prepare glass needles by pulling pasta peppes over a flame.
After sharpening the fine extremity of the pipette under the stereo microscope, connect the pipette to an aspiration plastic tube. To begin the blood collection, cut the skin above the posterior foot of the anesthetized frog to expose the dorsal tarsus vein. Fill the glass needle with one to two milliliters of ice cold A PBS plus heparin solution.
Then insert the glass needle into the vein and start collecting blood by slowly aspirating with the mouth. One to two milliliters of blood can be obtained from one average sized frog. The small incision on the frog's leg does not need to be sewed.
The wound will heal within a week. Results of a stereo microscopic analysis of skin graft rejection 12 days post transplantation are shown here. An LG six cloned frog that received a skin graft from an MHC identical LG six donor showed no rejection.
The asterisk indicates the marker forceps not due to rejection. In contrast, an LG six cloned frog that received a skin graft from an MHC disparate outbred donor shows 80%rejection. In both images, arrows indicates silvery irid four pigmented cells marking healthy non rejected grafted tissue.
In this next experiment, LG 15 pls were pulsed with either a PBS as a negative control recombinant GP 96 purified from an e coli culture or GP 96 purified from 15 zero tumor tissue cells were then adoptively transferred into LG 15 adult recipients and three days later live 15 zero tumor cells were transplanted by subcutaneous injection tumors that first appeared days post challenge were monitored and tumor size was measured periodically in these graphs, each curve represents the kinetics of tumor growth in one frog. The results show that animals primed with either 0.5 or one microgram of GP 96 had significantly delayed tumor appearance in comparison to the control groups. Therefore, GP 96 facilitates cross presentation of tumor antigens in xenopus.
So we have just shown you how to explore the ability of GP 96 to represent both minor and tumor antigens in the frog xap lavis by skin grafting and tumor transplantation assays. When doing this procedure, it is important to remember to avoid collecting peritoneal lavage if a blood vessel has been damaged since the majority of cells within erythrocytes and not macrophages. It is also important to avoid squeezing the tissue with the forceps when manipulating the skin graft in order to avoid damage.
So that's it. Thanks for washing and good luck with your experiments.
