This protocol can provide other researchers with direct and effective experimental approaches and the detail steps to evaluate the cellular response efficacies of novel vaccine adjuvants. It cannot only provide researchers with specific cellular evaluation methods for adjuvants, but can also break down extraction methods such as BM disease. This protocols not only demonstrate no toxicity and high cellular delivery, but also provide detailed procedures for adjuvant evaluation field.
To begin, turn on the water bath and adjust the temperature to 37 degrees Celsius. Then after collecting a tube of frozen L929 cells from liquid nitrogen, thaw quickly in the water bath. After pipetting the cells into a 15 milliliters sterile centrifuge tube, add two milliliters of DMEM and mix well.
Centrifuge the samples at 129 x g for five minutes. And after discarding the supernatant, add two milliliters of DMEM to resuspend the cells. Centrifuge the samples again.
And after discarding the supernatant, add six milliliters of DMEM complete medium for resuspension, and transferred to a T25 centimeter squared culture flask in a 37 degree Celsius incubator with 5%carbon dioxide to culture for 48 hours. On day 24 after the primary immunization, remove the mice from the animal room, place it in a glass dish and soak in 75%alcohol for five minutes. Place the centrifuge tubes on a centrifuge tube rack, numbered the disposable Petri dishes, and add five milliliters of PBS to each Petri dish with a 10 milliliter pipette.
Make a six to eight centimeter incision with scissors in the middle of the left ventral side of the mouse. Tear open the skin to expose the abdominal wall and locate the long red strip of the spleen. Next, lift the peritoneum on the inferior side of the spleen with forceps.
Cut it open and turn it upward to expose the spleen. Lift the spleen with forceps. Separate the connective tissue beneath the spleen with ophthalmic scissors and remove the spleen.
Place the spleen in a Petri dish containing five milliliters of PBS and mill with a sieve and syringe plunger. After grinding, transfer the liquid into a 15 milliliter centrifuge tube with a pipette in accordance with the numbering. Next, centrifuge the liquid at 453 x g for five minutes.
And after discarding the supernatant, add three milliliters of red blood cell lysis buffer to each tube. Resuspend the cells and lyse at room temperature for 10 minutes. Then add 10 to 12 milliliters of PBS to each tube.
And after mixing and centrifuging at 453 x g for five minutes, discard the supernatant and add 10 milliliters of PBS to each centrifuge tube and resuspend the cells. Take 20 microliters of each sample in a well of the cell counting plate and record the number of live cells using an automated cell counter. Next, centrifuge the samples at 453 x g for five minutes.
And after discarding the supernatant, dilute to 2.5 x 10 to the sixth cells per milliliter with RF10 medium and add to a 96-well plate at 100 microliters per well. Cut a six to eight centimeters incision below the abdomen of the mouse with scissors and clamp the two ends of the opening to separate it in different directions and expose the legs of the mouse. Separate the mouse femur from the mouse body and the tibia from the joint.
And keep the bones intact at both ends. Next, remove the residual tissue and cartilage from the articular joints at both ends of the femur with scissors and forceps. Soak the femurs in 75%alcohol for five minutes and then soak in a sterile PBS solution to wash off the surface alcohol.
Then cut off the ends of the femurs with scissors and rinse the bone marrow in a sterile Petri dish with sterile PBS solution, followed by aspiration with a one milliliter syringe. Repeat the washing three to five times. Next, filter by a cell sieve and collect the bone marrow-derived dendritic cells into a 15 milliliter centrifuge tube.
After centrifuging the samples at 290 x g for five minutes, discard the supernatant and add four milliliters of red blood cell lysis buffer, resuspend and lyse at room temperature for five minutes. Next, add 10 milliliters of sterile PBS solution to neutralize the lysate, and after centrifuging at 290 x g for five minutes, discard the supernatant. Resuspend the cells in one milliliter of DMEM containing 1%penicillin-streptomycin solution and 10%fetal bovine serum and count.
Then add granulocyte macrophage colony stimulating factor plus interleukin-4 to the medium. Adjust the cell concentration to 5 x 10 to the fifth per milliliter and inoculate the cells on cover slips. After adding the cell suspension to a six-well plate at two milliliters per well, place the plate in a humidified incubator at 37 degrees Celsius with 5%carbon dioxide for 48 hours.
Change the medium completely after two days and change half of the medium after four days. Immerse the mice in 75%alcohol after euthanasia and place them face up in a glass Petri dish in numbered order. Pass the mice through the transfer window into the sterile operation room and place them on the operation table for five minutes.
Using the syringe, aspirate 10 milliliters of saline. Tilt the mice downward at approximately 45 degrees and inject into the middle of the abdominal cavity. Draw about five milliliters of cell suspension into a 15 milliliter centrifuge tube for each 10 milliliters and repeat the injection three times.
Centrifuge the cell suspension at 129 x g for five minutes to obtain mouse peritoneal primary macrophages. L929 fibroblasts are a useful screening model for the in vitro toxicity testing of NOD. The quantification of inflammatory cytokine levels in the spleen can help researchers better understand the immune response.
Monitoring cytotoxic T lymphocytes with enzyme-linked immunospot is the gold standard for assessing antigen specific T-cell immunity in clinical trials and for screening vaccine candidates. The increased uptake of an antigen by dendritic cells can elicit enhanced adaptive immune responses. Macrophages play an important role in presenting antigens to T-cells, as well as inducing other antigen presenting cells to express co-stimulatory molecules, thereby initiating adaptive immune responses.
The isolation of cells under the ratio of drug to cell action are of utmost importance.
