In Vitro Cellular Activity Evaluation of the Nanoemulsion Vaccine Adjuvant Ophiopogonin D

All cell experiments were performed in a cell engineering laboratory equipped with basic operating rooms, buffer rooms, sterile culture rooms, and identification and analysis rooms. The working environment and conditions were free from microbial contamination and other harmful factors. The animal experiments were conducted based on the Guidelines for the Care and Use of Laboratory Animals and were approved by the Laboratory Animal Welfare and Ethics Committee of the Third Military Medical University.

1. Autoclaving and material preparation

1. Prepare the reagents and consumables, such as phosphate-buffered saline (PBS), scissors, forceps, and abrasive mesh, by moist heat sterilization by autoclaving at 121 °C for 20 min.
2. For the required reagents and equipment, see the Table of Materials. For the formula of the blank nanoemulsion (BNE), check Table 1.

2. L929 cytotoxicity assay

1. Turn on the water bath and adjust the temperature to 37 °C. Collect one tube of frozen L929 cells from liquid nitrogen and thaw quickly in a 37 °C water bath.
2. Pipette the cells into a 15 mL sterile centrifuge tube quickly after thawing, add 2 mL of DMEM, and mix well.
3. Centrifuge the samples at 129 x g for 5 min and discard the supernatant. Then, add 2mL of DMEM to resuspend the cells, and centrifuge the samples again at 129 x g for 5 min.
4. Discard the supernatant, add 6 mL of DMEM complete medium (containing 10% FBS) for resuspension, and transfer to a T25 culture flask in a 37 °C incubator with 5% CO₂ to culture for 48 h.
5. Discard the culture medium in the culture flask and wash the cells twice with 2 mL of PBS. Then, add 1 mL of 0.25% trypsin to digest the cells for 1-2 min at 37 °C.
6. When rounding of the cells is observed, add 4 mL of DMEM complete medium to terminate digestion immediately and mix well. Then, aspirate the cells into a 15 mL sterile centrifuge tube and centrifuge at 129 x g for 5 min.
7. Remove the supernatant and resuspend the cells in 1 mL of DMEM complete medium. Use a 20 µL cell suspension for cell counting using cell counting plates and dilute the remaining cells to 1 x 10⁵ cells/mL with DMEM complete medium.
8. Add 100 µL of ultrapure water to the periphery of a 96-well plate and add 100 µL of cell diluent to the internal wells only. Place the plate in the incubator for adherent culture for 4 h at 37 °C.
9. After the cells adhere, add OP-D and NOD separately in DMEM complete medium to a final volume of 200 µL/well (the final concentrations of each drug are 480 µg/mL, 240 µg/mL, 120 µg/mL, 60 µg/mL, and 30 µg/mL). For each concentration, use three wells as replicates. Then, place the cells back into the incubator and culture for another 24 h.
10. Dilute CCK-8 to 10% with DMEM complete medium and add 100 µL/well dilutions containing the adjuvant and cell solutions to the 96-well plate. Place the plate back into the incubator and incubate for another 2-3 h.
11. Mix frequently while plating the cell solution to prevent inhomogeneity due to cell precipitation. Gently shake the plate several times before and after adding the CCK-8 to mix the medium and CCK-8 solution well.
12. Measure the absorbance at 450 nm using a microplate reader. Set up a zeroing well with only medium and CCK-8 for a baseline absorbance value. Calculate accurate absorbance values by subtracting the zeroed absorbance value from the obtained absorbance value when plotting.
13. Confirm that no bubbles are present in any wells before testing with the microplate reader because bubbles will interfere with the assay.

3. Splenocyte stimulation

1. Immunize BALB/c mice aged 6-8 weeks with 30 µg of protein antigen plus 30 µg of adjuvant via an intramuscular injection (200 µL) on day 0, day 7, and day 14 according to the following experimental groups: (1) PBS group, (2) antigen (Ag) group, (3) antigen + OP-D (Ag/OP-D) group, (4) antigen + BNE (Ag/BNE) group, (5) antigen + NOD (Ag/NOD) group, and (6) antigen + AlPO₄ (Ag/Al) group.
2. Preparation room: On day 24 after the primary immunization, remove the mice from the animal room and euthanize them by an intraperitoneal injection of 100 mg/kg of 1% sodium pentobarbital. Place the mice in a glass dish and soak in 75% alcohol for 5 min.
3. Place the centrifuge tubes on a centrifuge tube rack, number the disposable Petri dishes, and add 5 mL of PBS to each Petri dish with a 10 mL pipette.
4. Make a 6-8 cm incision with scissors in the middle of the left ventral side of the mouse, tear open the skin, expose the abdominal wall, and locate the long red strip of the spleen.
5. 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.
6. Place the spleen in a Petri dish containing 5 mL of PBS and mill with a sieve (200 mesh, 70 µm) and grinding bar. After grinding, transfer the liquid into a 15 mL centrifuge tube with an elbow dropper in accordance with the numbering.
7. Centrifuge the liquid at 453 x g for 5 min. Discard the supernatant, add 3 mL of red blood cell lysis buffer to each tube, resuspend the cells, and lyse at room temperature for 10 min.
8. Add 10-12 mL of PBS to each tube, mix the tube upside down, and centrifuge at 453 x g for 5 min. Discard the supernatant, add 10 mL of PBS to each centrifuge tube, and resuspend the cells.
9. Take 20 µL of each sample in a well of the cell counting plate and record the number of live cells using an automated cell counter.
10. Centrifuge the samples at 453 x g for 5 min and discard the supernatant. Resuspend the cells, dilute to 2.5 x 10⁶ cells/mL with RF-10 medium (formulation information in Table 2), and add to a 96-well plate at 100 µL/well.
11. Dilute the antigen with RF-10 medium to 10 µg/mL, add 100 µL to each well, and incubate for 3 days at 37 °C in 5% CO₂.
12. Aspirate the cell suspension obtained from each group of cells in 1.5 mL centrifuge tubes, centrifuge at 453 x g for 20 min, and aspirate the supernatant into a clean centrifuge tube.
13. Carry out IFN-γ and IL-17A content detection strictly according to the ELISA kit instructions. The methods and procedures are as follows.
14. Prepare 1x washing buffer working solution (provided with the kit), standard gradient concentration solution (dilute cytokine standard solution to 500 pg/mL, 250 pg/mL, 125 pg/mL, 62.5 pg/mL, 31.3 pg/mL, and 15.6 pg/mL in the dilution buffer R [1x] provided with the kit), biotinylated antibody working solution (dilute biotinylated antibody solution to 1:100 using the dilution buffer R [1x] provided with the kit to form the working solution), and streptavidin-HRP working solution as needed.
15. Add 100 µL/well of diluted cytokine standard solution into the standard well, 100 µL/well of sample into the sample well (the dilution buffer R [1x] provided with the kit is used for the sample dilution), and 100 µL/well of dilution buffer R (1x) into the blank control well.
16. Add biotinylated antibody working solution at 50 µL/well. Mix well, cover with a sealing membrane, and incubate at 37 °C for 90 min.
17. Remove the liquid from the wells and add 1x washing buffer working solution at 300 µL/well. Discard the liquid from the wells after 1 min. Repeat this process 4x allowing the liquid to dry on filter paper each time.
18. Add 100 µL/well of streptavidin-HRP working solution. Cover and incubate the samples at 37 °C for 30 min. Centrifuge the samples at 453 x g for 5 min and discard the supernatant.
    NOTE: The washing solution remaining in the reaction well during the washing process should be thoroughly patted until no watermark can be seen on the filter paper.
19. Add TMB at 100 µL/well, incubate the plates at 37 °C for 5-30 min in the dark, and judge the termination reaction according to the depth of color in the well (dark blue). Usually, 10-20 min for color development can achieve good results.
20. Terminate the reaction quickly by adding stop solution at 100 µL/well. Detect the absorbance value at 450 nm within 10 min after termination.
    ​NOTE: Equilibrate the reagent at room temperature for 30 min before use.

4. ELISpot assay

1. Perform immunization of the mice and the collection of splenocytes exactly as described in steps 3.1-3.10 above. Perform the assays for IFN-γ and IL-17A in strict accordance with the kit instructions. The methods and procedures are as follows.
2. Remove the plate from the sealed package, wash 4x with sterile PBS (200 µL/well), and add 1640 complete culture medium (200 µL/well) to balance it at room temperature for 2 h.
3. Remove the medium and dilute the splenocyte suspension with RF-10 medium to 2 x 10⁵ cells/mL, adding 50 µL of cells and antigen per well (final concentration: 10 µg/mL).
4. Place the plate in a humidified incubator at 37 °C with 5% CO₂ for 48 h. Do not move the plate during this time, and take measures to avoid evaporation (e.g., wrap the plate with aluminum foil).
5. Dilute the detection antibody (BVD6-24G2-biotin) to 1 µg/mL (1:1,000) in phosphate buffered saline (0.5 mL:100 mL) containing 0.5% fetal bovine serum (PBS-0.5% FBS). Add 100 µL/well to the plate, and incubate it at room temperature for 2 h after filtration through a 0.22 µm membrane.
6. Decant the liquid in the wells, add 1x washing buffer at 200 µL/well, and wash 5x. Leave for 30-60 s each time, and for the last time, allow to dry on blotting paper.
7. Dilute the streptavidin-horseradish peroxidase (1:1,000) in PBS-0.5% FBS and add 100 µL/well. Incubate the plate for 1 h at room temperature. Wash the plate as in step 4.6.
8. Add 100 µL/well of ready-to-use TMB substrate solution and develop until a clear spot appears. Stop the color development by washing extensively in deionized water (rinse 5x-6x repeatedly). If necessary, remove the culvert (the soft plastic under the plate), and rinse the lower side of the membrane.
9. Examine and count the spots on an ELISpot reader or dissecting microscope after the plate has dried.

5. Uptake by DCs

1. Euthanize BALB/c normal mice by an intraperitoneal injection of 100 mg/kg of 1% sodium pentobarbital. Soak the mice in 75% alcohol for 5 min.
2. Cut a 6-8 cm 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.
3. 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 5 min, and then soak in sterile PBS solution to wash off the surface alcohol.
4. 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 1 mL syringe. Repeat the washing 3x-5x.
5. Filter by a cell sieve (200 mesh, 70 µm) and collect the BMDCs into a 15 mL centrifuge tube. Centrifuge the samples at 290 x g for 5 min. Discard the supernatant, add 4 mL of red blood cell lysis buffer, resuspend, and lyse at room temperature for 5 min.
6. Add 10 mL of sterile PBS solution to neutralize the lysate, centrifuge at 290 x g for 5 min, and discard the supernatant.
7. Resuspend the cells in 1 mL of DMEM containing 1% penicillin-streptomycin solution and 10% FBS and count. Then, add GM-CSF (20 ng/mL) plus IL-4 (10 ng/mL) to the medium, adjust the cell concentration to 5 x 10⁵/mL, and inoculate the cells on coverslips.
8. Add the cell suspension to a 6-well plate at 2 mL/well, and place the plate in a humidified incubator at 37 °C with 5% CO₂ for 48 h. Change the medium completely after 2 days, and change half of the medium after 4 days.
9. Select five wells with cells in good condition (large and radiolucent cells with dendritic protrusions on the cell surface) using an inverted microscope at 100x magnification for the experiment on the seventh day of culture. Discard the supernatant, add 2 mL of GFP, OP-D + GFP, and NOD + GFP solutions diluted with DMEM complete medium (GFP final concentration: 20 µg/mL; adjuvant final concentration: 10 µg/mL) to each well, and incubate the plates at 37 °C for 30 min in the dark.
10. Wash the plates 3x with PBS, add 1 mL of 4% paraformaldehyde to each well, and incubate at room temperature for 15 min. Remove the paraformaldehyde after fixation, incubate the cells with phalloidin and DAPI to a final concentration of 10 µg/mL for 10 min for staining, and then wash 3x with PBS.
11. Add 1 mL of PBS to each well, and observe the antigen uptake using CLSM, as described below.
    1. Open the CLSM software, click on ZEN System, and wait for the hardware initialization to complete.
    2. Click on the GFP and DAPI shortcuts in locate tab to find the area that can be observed. Turn off the fluorescent light and transmitted light.
    3. Click on Smart Setup in acquisition menu to open the dye library, and add three fluorescent dyes: EGFP, phalloidin, and DAPI. Click on Best Signal > OK. Click on the EGFP channel in channels tab and click on Live to select the correct field of view on the right interface.
    4. Select 1 AU for the pinhole, rotate the fine focusing screw to adjust the focal length, and select the appropriate focal plane. Click on Range Indicator and adjust the combination of laser power and master gain so that only sporadic red dots appear on the image.
    5. Adjust the phalloidin and DAPI channels with the same parameters without changing the laser power.
    6. Select Stop Live and click on Acquisition Mode to change the shooting parameters: Frame Size: 1024 pixels x 1024 pixels; Scan Speed: 7; Averaging: 2x. Click on Snap and select Split to view all the images taken.Save the images.

6. Macrophage activation

1. Immerse C57BL/6 mice in 75% alcohol after euthanasia and place them face up in a glass Petri dish in numbered order.
2. Pass the mice through the transfer window into the sterile operation room and place on the operation table for 5 min.
3. Using a syringe, aspirate 10 mL of saline, tilt the mice downward at approximately 45°, and inject into the middle of the abdominal cavity. Draw about 5 mL of cell suspension into a 15 mL centrifuge tube for each 10 mL and repeat the injection 3x.
4. Centrifuge the cell suspension at 129 x g for 5 min to obtain mouse peritoneal primary macrophages. Resuspend the cells, adjust the cell concentration to 2 x 10⁶ cells/mL with RPMI 1640 complete medium (containing 10% FBS), and inoculate the cell suspension in a 24-well plate to ensure a consistent number of cells per well (1 mL/well).
5. Culture at 37 °C in 5% CO₂ overnight (approximately 16-20 h), followed by incubation with PBS, Ag, Ag/OP-D, Ag/NOD, and Ag/Al (Ag final concentration: 5 µg/mL; adjuvant final concentration: 10 µg/mL; total volume: 2 mL) for 24 h. Detect the levels of IL-1β, IL-6, and TNF-α in the culture supernatant with ELISA kits using the methods and procedures described in steps 3.12-3.19.