These protocols provide an effective way to study the antitumor activity of immune modulator attacks.Moreover, the analysis of the associated chances in the circulating cytokines and immune cell populations can be done using these proposed methods.The main advantage of this protocol is it's easy and a straight forward way of implanting a tumor and monitor its growth.The direct implication of this technique is to assess the efficacy of certain therapies or challenges in anti-cancer research.Additionally, some aspects of these techniques are suitable for toxicity studies.To begin, thaw a frozen vial of murine EERL cells in a preheated water bath at 37 degrees Celsius and transfer them to a 15 milliliter conical tube containing warm culture media.Centrifuge the conical tube at 277 times G for five minutes at 25 degrees Celsius to remove the media.Then re-suspend the cell palate in three to five milliliters fresh media and transfer it to a T25 cell culture flask.Let the cells grow in a humidified incubator at 37 degrees Celsius and 5%carbon dioxide, Expand to larger flasks and passage every three days.When there are enough cells for the desired implantation into all mice, remove the flasks, discard the media and gently rinse the cells PBS.Then add four milliliters of 0.25%trypsin EDTA and incubate at 37 degrees Celsius for two minutes.Add fresh media to stop the trypsin reaction and collect the cell suspension in 50 milliliter conical tubes.Re-suspend the cells in fresh media and count the cells.Centrifuge once more, then add cold PBS to the cells to make a final concentration of 10 million cells per milliliter.Keep the cell suspension on ice before injection.Disinfect the flank area with an ethanol pad.To inject cell suspension into mice, aspirate 100 microliters of cell suspension into the syringe and remove all the bubbles and dead space from the top.Then inject the cell suspension subcutaneously in a slow and steady manner into anesthetized mice.Place the animals in their respective cages and monitor them until they recover from anesthesia.To collect blood, grasp the loose skin over the shoulder using the non-dominant hand and puncture the submandibular vein with an 18 gauge needle or lancet.Collect 200 to 300 microliters of blood into a 1.5 milliliter polypropylene micro centrifuge tube or serum separator tube.After blood collection, apply gentle pressure to the puncture site until the bleeding has stopped.Return the mice to their respective cages and observe until they recover from anesthesia.Let the collected blood to clot at room temperature for 20 to 30 minutes.Then place the tubes on ice until they are ready to be centrifuged.Centrifuge the clotted blood at 1, 540 times G for 15 minutes at four degrees Celsius, and carefully collect the serum layer from the top.Thaw the serum or plasma samples while keeping them on ice.Then, centrifuge the samples at 1, 540 times G for five minutes at four degrees Celsius, to sediment any cell debris and carefully collect the serum layer from the top.Place the multiplex kit at room temperature and perform the assay according to the manufacturer's protocol.Lay each mouse on its back and spray 70%ethanol on the abdominal area skin.Use forceps and scissors to cut the lymph node from the right side of the mice and the tumor out from the left side.If the tumor is big, cut it into small pieces and take about 500 to 600 milligrams off the tissue.Place the tissues onto the respective dissociator tubes containing three to five milliliters of RPMI media.Homogenize the tissue using an automated dissociator.After homogenization, filter the cell suspension through a 70 micrometer filter into a 50 milliliter conical tube.Centrifuge at 277 times G for five minutes at 25 degrees Celsius to remove the media.Wash and re-suspend the cells in one to two milliliter of cold PBS.Use 0.4%trypsin blue staining, to count the viable cells in a hemocytometer.Calculate the volume required to get two to three million cells and transfer them to the respective fax tube.Centrifuge counted cells at 277 times G for five minutes at 25 degrees Celsius.Decant the supernatant and re-suspend the cells into 300 microliters of PBS.Add 0.5 to one microliter of zombie dye per tube and incubate at room temperature for 20 minutes in the dark.Centrifuge the cells Wash them with one to two milliliters of fax buffer and re-suspend them into 200 microliters of fax buffer.Add FC receptor blocker according to the manufacturer's protocol and incubate the cells for 10 minutes.Centrifuge again at previously described settings and wash the cells with one to two milliliters of fax buffer.Add the antibody cocktail and incubate for 30 minutes at four degrees Celsius in the dark.After the incubation, centrifuge and wash the cells with one to two milliliters fax buffer.Add 300 to 400 microliters of 2%paraformaldehyde solution, and re-suspend for fixation.Store the cells for a couple of days at four degrees Celsius or analyze immediately by multicolor flow cytometer.In this study, the antitumor activity of polyannhydride IL-1
