This study was supported by grant No. 2021YFC2302603 of the National Key Research and Development Program of China, grants No. 31670938, 32070924, 82041045, and 32000651 of the National Natural Science Foundation Program of China, grants No. 2014jcyjA0107 and No. 2019jcyjA-msxmx0159 of the Natural Science Foundation Project Program of Chongqing, grant No. CYS21519 of the Postgraduate Research and Innovation Project of Chongqing, grant No. 2020XBK24 of the Army Medical University Special projects, and grant No. 202090031021 of the National Innovation and Entrepreneurship Program for college students.

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
<ol>
	<li>Prepare the reagents and consumables, such as phosphate-buffered saline (PBS), scissors, forceps, and abrasive mesh, by moist heat sterilization by autoclaving at 121 &#176;C for 20 min.</li>
	<li>For the required reagents and equipment, see the Table of Materials. For the formula of the blank nanoemulsion (BNE), check Table 1.</li>
</ol>
2. L929 cytotoxicity assay
<ol>
	<li>Turn on the water bath and adjust the temperature to 37 &#176;C. Collect one tube of frozen L929 cells from liquid nitrogen and thaw quickly in a 37 &#176;C water bath.</li>
	<li>Pipette the cells into a 15 mL sterile centrifuge tube quickly after thawing, add 2 mL of DMEM, and mix well.</li>
	<li>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.</li>
	<li>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 &#176;C incubator with 5% CO2 to culture for 48 h.</li>
	<li>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 &#176;C.</li>
	<li>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.</li>
	<li>Remove the supernatant and resuspend the cells in 1 mL of DMEM complete medium. Use a 20 &#181;L cell suspension for cell counting using cell counting plates and dilute the remaining cells to 1 x 105 cells/mL with DMEM complete medium.</li>
	<li>Add 100 &#181;L of ultrapure water to the periphery of a 96-well plate and add 100 &#181;L of cell diluent to the internal wells only. Place the plate in the incubator for adherent culture for 4 h at 37 &#176;C.</li>
	<li>After the cells adhere, add OP-D and NOD separately in DMEM complete medium to a final volume of 200 &#181;L/well (the final concentrations of each drug are 480 &#181;g/mL, 240 &#181;g/mL, 120 &#181;g/mL, 60 &#181;g/mL, and 30 &#181;g/mL). For each concentration, use three wells as replicates. Then, place the cells back into the incubator and culture for another 24 h.</li>
	<li>Dilute CCK-8 to 10% with DMEM complete medium and add 100 &#181;L/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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Confirm that no bubbles are present in any wells before testing with the microplate reader because bubbles will interfere with the assay.</li>
</ol>
3. Splenocyte stimulation
<ol>
	<li>Immunize BALB/c mice aged 6-8 weeks with 30 &#181;g of protein antigen plus 30 &#181;g of adjuvant via an intramuscular injection (200 &#181;L) 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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Place the spleen in a Petri dish containing 5 mL of PBS and mill with a sieve (200 mesh, 70 &#181;m) and grinding bar. After grinding, transfer the liquid into a 15 mL centrifuge tube with an elbow dropper in accordance with the numbering.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Take 20 &#181;L of each sample in a well of the cell counting plate and record the number of live cells using an automated cell counter.</li>
	<li>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 &#181;L/well.</li>
	<li>Dilute the antigen with RF-10 medium to 10 &#181;g/mL, add 100 &#181;L to each well, and incubate for 3 days at 37 &#176;C in 5% CO2.</li>
	<li>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.</li>
	<li>Carry out IFN-&#947; and IL-17A content detection strictly according to the ELISA kit instructions. The methods and procedures are as follows.</li>
	<li>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.</li>
	<li>Add 100 &#181;L/well of diluted cytokine standard solution into the standard well, 100 &#181;L/well of sample into the sample well (the dilution buffer R [1x] provided with the kit is used for the sample dilution), and 100 &#181;L/well of dilution buffer R (1x) into the blank control well.</li>
	<li>Add biotinylated antibody working solution at 50 &#181;L/well. Mix well, cover with a sealing membrane, and incubate at 37 &#176;C for 90 min.</li>
	<li>Remove the liquid from the wells and add 1x washing buffer working solution at 300 &#181;L/well. Discard the liquid from the wells after 1 min. Repeat this process 4x allowing the liquid to dry on filter paper each time.</li>
	<li>Add 100 &#181;L/well of streptavidin-HRP working solution. Cover and incubate the samples at 37 &#176;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.</li>
	<li>Add TMB at 100 &#181;L/well, incubate the plates at 37 &#176;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.</li>
	<li>Terminate the reaction quickly by adding stop solution at 100 &#181;L/well. Detect the absorbance value at 450 nm within 10 min after termination. 
	&#8203;NOTE: Equilibrate the reagent at room temperature for 30 min before use.</li>
</ol>
4. ELISpot assay
<ol>
	<li>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-&#947; and IL-17A in strict accordance with the kit instructions. The methods and procedures are as follows.</li>
	<li>Remove the plate from the sealed package, wash 4x with sterile PBS (200 &#181;L/well), and add 1640 complete culture medium (200 &#181;L/well) to balance it at room temperature for 2 h.</li>
	<li>Remove the medium and dilute the splenocyte suspension with RF-10 medium to 2 x 105 cells/mL, adding 50 &#181;L of cells and antigen per well (final concentration: 10 &#181;g/mL).</li>
	<li>Place the plate in a humidified incubator at 37 &#176;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).</li>
	<li>Dilute the detection antibody (BVD6-24G2-biotin) to 1 &#181;g/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 &#181;L/well to the plate, and incubate it at room temperature for 2 h after filtration through a 0.22 &#181;m membrane.</li>
	<li>Decant the liquid in the wells, add 1x washing buffer at 200 &#181;L/well, and wash 5x. Leave for 30-60 s each time, and for the last time, allow to dry on blotting paper.</li>
	<li>Dilute the streptavidin-horseradish peroxidase (1:1,000) in PBS-0.5% FBS and add 100 &#181;L/well. Incubate the plate for 1 h at room temperature. Wash the plate as in step 4.6.</li>
	<li>Add 100 &#181;L/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.</li>
	<li>Examine and count the spots on an ELISpot reader or dissecting microscope after the plate has dried.</li>
</ol>
5. Uptake by DCs
<ol>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Filter by a cell sieve (200 mesh, 70 &#181;m) 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.</li>
	<li>Add 10 mL of sterile PBS solution to neutralize the lysate, centrifuge at 290 x g for 5 min, and discard the supernatant.</li>
	<li>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.</li>
	<li>Add the cell suspension to a 6-well plate at 2 mL/well, and place the plate in a humidified incubator at 37 &#176;C with 5% CO2 for 48 h. Change the medium completely after 2 days, and change half of the medium after 4 days.</li>
	<li>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 &#181;g/mL; adjuvant final concentration: 10 &#181;g/mL) to each well, and incubate the plates at 37 &#176;C for 30 min in the dark.</li>
	<li>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 &#181;g/mL for 10 min for staining, and then wash 3x with PBS.</li>
	<li>Add 1 mL of PBS to each well, and observe the antigen uptake using CLSM, as described below.
	<ol>
		<li>Open the CLSM software, click on ZEN System, and wait for the hardware initialization to complete.</li>
		<li>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.</li>
		<li>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 &#62; OK. Click on the EGFP channel in channels tab and click on Live to select the correct field of view on the right interface.</li>
		<li>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.</li>
		<li>Adjust the phalloidin and DAPI channels with the same parameters without changing the laser power.</li>
		<li>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.</li>
	</ol>
	</li>
</ol>
6. Macrophage activation
<ol>
	<li>Immerse C57BL/6 mice in 75% alcohol after euthanasia and place them face up in a glass Petri dish in numbered order.</li>
	<li>Pass the mice through the transfer window into the sterile operation room and place on the operation table for 5 min.</li>
	<li>Using a syringe, aspirate 10 mL of saline, tilt the mice downward at approximately 45&#176;, 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.</li>
	<li>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).</li>
	<li>Culture at 37 &#176;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 &#181;g/mL; adjuvant final concentration: 10 &#181;g/mL; total volume: 2 mL) for 24 h. Detect the levels of IL-1&#946;, IL-6, and TNF-&#945; in the culture supernatant with ELISA kits using the methods and procedures described in steps 3.12-3.19.</li>
</ol>

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M., Williams, A., Eisenbarth, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+and+function+of+the+immune+system+in+the+spleen.">Structure and function of the immune system in the spleen.</a> Science Immunology. 4 (33), (2019).</li><li>Cox, J. H., Ferrari, G., Janetzki, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+cytokine+release+at+the+single+cell+level+using+the+ELISPOT+assay.">Measurement of cytokine release at the single cell level using the ELISPOT assay.</a> Methods. 38 (4), 274-282 (2006).</li><li>Elliott, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Confocal+microscopy:+Principles+and+modern+practices.">Confocal microscopy: Principles and modern practices.</a> Current Protocols in Cytometry. 92 (1), 68 (2020).</li><li>Zhou, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD4(+)+T+cell+activation+and+inflammation+in+NASH-related+fibrosis.">CD4(+) T cell activation and inflammation in NASH-related fibrosis.</a> Frontiers in Immunology. 13, 967410 (2022).</li><li>Martinez, F. O., Sica, A., Mantovani, A., Locati, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macrophage+activation+and+polarization.">Macrophage activation and polarization.</a> Frontiers in Bioscience. 13, 453-461 (2008).</li><li>Quesniaux, V., Erard, F., Ryffel, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adjuvant+activity+on+murine+and+human+macrophages.">Adjuvant activity on murine and human macrophages.</a> Methods in Molecular Biology. 626, 117-130 (2010).</li></ol>

The authors declare that there are no competing financial or personal interests that could have influenced the work reported in this paper.

Subunit vaccines provide excellent safety but poor immunogenicity. The main strategy to enhance the immunogenicity is to physically adsorb or couple antigens with adjuvants and incorporate them into the drug delivery systems to promote the uptake and presentation by DCs. Natural plant saponins such as quillaia saponin and its derivatives are highly toxic and are not suitable for the development of human vaccines17. Therefore, the study of the toxic effects of vaccines or adjuvants on cells is a ne...

Vaccines are an important means of preventing and treating infectious and noncommunicable diseases. The appropriate addition of adjuvants and delivery vehicles to vaccine formulations is beneficial for enhancing the immunogenicity of antigens and generating long-lasting immune responses1. In addition to the classical adjuvant alum (aluminum salt), there are six kinds of adjuvants for vaccines that are currently marketed: MF592,3, AS043, AS033, AS013, CpG10184, and Matrix-M5. Generally, when the human body encounters a viral attack, the first and second lines of defense (skin, mucosa, and macrophages) take the lead in clearing the virus, and finally, the third line of defense, involving the immune organs and immune cells, is activated. Aluminum and aluminum salts have been the most widely used adjuvants for human vaccines since the early 1920s, eliciting an effective innate immune response6. However, it has been proposed that the activation of antigen-presenting cells (APCs) by classical adjuvants, which stimulates the immune cells to generate specific sets of cytokines and chemokines, is the mechanism by which adjuvants work and may be one of the reasons why adjuvants exert only transient effects on specific immune responses7. The presence of limited licensed adjuvants for human use is a restrictive factor for developing vaccines that elicit effective immune responses8.
Currently, an increasing number of adjuvant studies are demonstrating the ability to induce a strong cellular immune response in mice. QS-21 has been shown to induce a balanced T-helper 1 (Th1) and T-helper 2 (Th2) immune response, produce higher levels of antibody titers, and prolong the protection as an adjuvant, but its strong toxicity and hemolytic properties limit its development as a standalone clinical adjuvant9,10. OP-D (ruscogenin-O-&#945;-L-rhamnopyranosy1-(1&#8594;2)-&#946;-D-xylopyranosyl-(1&#8594;3)-&#946;-D-fucopyranoside) is one of the steroidal saponins isolated from the root of the Chinese medicinal plant Ophiopogon japonicas4. Additionally, it is the chief pharmacologically active component (Shen Mai San) found in Radix Ophiopogonis and is known to have certain pharmacological properties11. Moreover, it is a member of the Liliaceae family and is widely utilized for its inhibitory and protective effects in cellular inflammation and myocardial injury. For example, OP-D ameliorates DNCB-induced atopic dermatitis-like lesions and tumor necrosis factor alpha (TNF-&#945;) inflammatory HaCaT cells in BALB/c mice12. Importantly, OP-D promotes the antioxidative protection of the cardiovascular system and protects the heart against doxorubicin-induced autophagic injury by reducing both reactive oxygen species generation and disrupting mitochondrial membrane damage. Experiments have shown that taking OP-D with mono-desmoside helps to boost immune health, increase white blood cell counts and DNA synthesis, and make antibodies last longer13. It has previously been found that OP-D has an adjuvant effect14.
Nanoemulsions are oil-in-water nanoformulations composed of a combination of surfactants, oil, cosurfactants, and water12,15. These nanovaccine designs allow antigens and adjuvants to be encapsulated together to enhance immune stimulation, protect the antigens, and promote dendritic cell (DC) maturation16. For development of these novel adjuvants obtained from screening, it is important to find appropriate methods to evaluate their cellular response abilities.
The purpose of this protocol is to systematically evaluate whether adjuvants can enhance phagocytosis and the expression of immune cells in in vitro cell culture and to elaborate on the main experimental methods. The experiment is divided into four subsections: (1) the toxicity of OP-D and NOD to L929 cells is determined by the cell counting kit-8 (CCK-8) assay; (2) the cytokine levels of endocrine IFN-&#947; and IL-17A and the corresponding cell numbers in immunized mice are detected by splenocyte stimulation and ELISpot assays; (3) the antigen presentation ability of DCs after adjuvant stimulation is observed using confocal microscopy; and (4) the three kinds of cytokines, IL-1&#946;, IL-6, and TNF-&#945;, in the supernatants obtained from peritoneal macrophages (PMs) in normal mice cocultured with adjuvants are detected.

<table><tbody><tr><td>0.25% Trypsin-EDTA (1x)</td><td>GIBCO, USA</td><td>25200056</td><td></td></tr><tr><td>96-well filter plates</td><td>Millipore. Billerica, MA</td><td>CLS3922</td><td></td></tr><tr><td>AlPO4</td><td>General Chemical Company, USA</td><td>null</td><td></td></tr><tr><td>Automated Cell Counter</td><td>Countstar, China</td><td>IC1000</td><td></td></tr><tr><td>BALB/c mice and C57BL/6 mice</td><td>Beijing HFK Bioscience Co. Ltd</td><td>null</td><td></td></tr><tr><td>caprylic/capric triglyceride (GTCC)</td><td>Beijing Fengli Pharmaceutical Co. Ltd., Beijing, China</td><td>null</td><td></td></tr><tr><td>CCK-8 kits</td><td>Dojindo, Japan</td><td>CK04</td><td></td></tr><tr><td>Cell Counting Plate</td><td>Costar, Corning, USA</td><td>CO010101</td><td></td></tr><tr><td>Cell Sieve</td><td>biosharp, China</td><td>BS-70-CS</td><td></td></tr><tr><td>Centrifuge 5810 R</td><td>Eppendorf, Germany&amp;nbsp;</td><td>5811000398</td><td></td></tr><tr><td>DAPI</td><td>Sigma-Aldrich, St. Louis, USA</td><td>D9542</td><td></td></tr><tr><td>DMEM basic(1x) medium</td><td>GIBCO, USA</td><td>C11885500BT</td><td></td></tr><tr><td>DSZ5000X Inverted Microscope</td><td>Nikon,Japan</td><td>DSZ5000X</td><td></td></tr><tr><td>EL-35 (Cremophor-35)</td><td>Mumbai, India</td><td>null</td><td></td></tr><tr><td>ELISpot classic</td><td>AID, Germany</td><td>ELR06</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>GIBCO, USA</td><td>10099141C</td><td></td></tr><tr><td>Full-function Microplate Reader</td><td>Thermo Fisher Scientific, USA</td><td>VL0000D2</td><td></td></tr><tr><td>GFP</td><td>Sigma-Aldrich, St. Louis, USA</td><td>P42212</td><td></td></tr><tr><td>Glutamax</td><td>Invitrogen, USA</td><td>35050061</td><td></td></tr><tr><td>Granulocyte Macrophage Colony-Stimulating Factor</td><td>GM-CSF, R&amp;amp;D Systems, USA</td><td>315-03</td><td></td></tr><tr><td>HEPES</td><td>Invitrogen, USA</td><td>15630106</td><td></td></tr><tr><td>HF 90/240 Incubator</td><td>Heal Force, Switzerland</td><td>null</td><td></td></tr><tr><td>IL-4</td><td>PeproTech, USA</td><td>042149</td><td></td></tr><tr><td>L929 cell line</td><td>FENGHUISHENGWU, China</td><td>&amp;nbsp;NCTC clone 929 (RRID:CVCL_0462)</td><td></td></tr><tr><td>Laser Scanning Confocal Microscopy</td><td>Zeiss, Germany</td><td>LSM 980</td><td></td></tr><tr><td>MONTANE 85 PPI</td><td>SEPPIC, France</td><td>L12910</td><td></td></tr><tr><td>MONTANOX 80 PPI</td><td>SEPPIC, France</td><td>36372K</td><td></td></tr><tr><td>Mouse IFN-&amp;gamma; ELISA kit</td><td>Dakewe, China</td><td>1210002</td><td></td></tr><tr><td>Mouse IFN-&amp;gamma; precoated ELISPOT kit</td><td>Dakewe, China</td><td>DKW22-2000-096</td><td></td></tr><tr><td>Mouse IL-17A ELISA kit</td><td>Dakewe, China</td><td>1211702</td><td></td></tr><tr><td>Mouse IL-17A ELISpot&lt;sup&gt;PLUS&lt;/sup&gt; Kit</td><td>ebiosciences, USA</td><td>3521-4HPW-2</td><td></td></tr><tr><td>Mouse IL-1&amp;beta; ELISA kit</td><td>Dakewe, China</td><td>1210122</td><td></td></tr><tr><td>Mouse IL-6 ELISA kit</td><td>Dakewe, China</td><td>1210602</td><td></td></tr><tr><td>Mouse TNF-&amp;alpha; ELISA kit</td><td>Dakewe, China</td><td>1217202</td><td></td></tr><tr><td>Non-essential amino acids(100x)</td><td>Invitrogen, USA</td><td>11140050</td><td></td></tr><tr><td>Ophiopogonin-D</td><td>Chengdu Purui Technology Co. Ltd</td><td>945619-74-9</td><td></td></tr><tr><td>Penicillin-Streptomycin Solution</td><td>Invitrogen, USA</td><td>15070063</td><td></td></tr><tr><td>Phalloidin</td><td>Solarbio, China</td><td>CA1620</td><td></td></tr><tr><td>Phosphate Buffered Saline</td><td>ZSGB-BIO, China</td><td>ZLI-9062</td><td></td></tr><tr><td>Red Blood Cell Lysis Buffer</td><td>Solarbio, China</td><td>R1010</td><td></td></tr><tr><td>RPMI 1640 medium</td><td>Hyclone (Life Technology), USA</td><td>SH30809.01</td><td></td></tr><tr><td>Sodium pyruvate(100 mM)</td><td>Invitrogen, USA</td><td>11360070</td><td></td></tr><tr><td>Squalene</td><td>Sigma, USA</td><td>S3626</td><td></td></tr><tr><td>&amp;beta;- Mercaptoethanol</td><td>Invitrogen, USA</td><td>21985023</td><td></td></tr></tbody></table>

in vitro cellular activity evaluation of the nanoemulsion vaccine adjuvant ophiopogonin d

As a principal ingredient of vaccines, adjuvants can directly induce or enhance the powerful, widespread, innate, and adaptive immune responses associated with antigens. Ophiopogonin D (OP-D), a purified component extracted from the plant Ophiopogon japonicus, has been found to be useful as a vaccine adjuvant. The problems of the low solubility and toxicity of OP-D can be effectively overcome by using a low-energy emulsification method to prepare nanoemulsion ophiopogonin D (NOD). In this article, a series of in vitro protocols for cellular activity evaluation are examined. The cytotoxic effects of L929 were determined using a cell counting kit-8 assay. Then, the secreted cytokine levels and corresponding immune cell numbers after the stimulation and culture of splenocytes from immunized mice were detected by ELISA and ELISpot methods. In addition, the antigen uptake ability in bone marrow-derived dendritic cells (BMDCs), which were isolated from C57BL/6 mice and matured after incubation with GM-CSF plus IL-4, was observed by laser scanning confocal microscopy (CLSM). Importantly, macrophage activation was confirmed by measuring the levels of IL-1&#946;, IL-6, and tumor necrosis factor alpha (TNF-&#945;) cytokines by ELISA kits after coculturing peritoneal macrophages (PMs) from blank mice with the adjuvant for 24 h. It is hoped that this protocol will provide other researchers with direct and effective experimental approaches to evaluate the cellular response efficacies of novel vaccine adjuvants.

The cellular activity evaluation of the adjuvants OP-D and NOD was completed in vitro according to the protocol. L929 fibroblasts are a useful screening model for the in vitro toxicity testing of NOD (Figure 1). The quantification of inflammatory cytokine levels in the spleen can help researchers better understand the immune response (Figure 2). Monitoring CTLs with ELISpot is the gold standard for assessing antigen-specific T-cell immunity in ...

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

The protocol presents detailed methods for evaluating whether the nanoemulsion ophiopogonin D adjuvant promotes effective cellular immune responses.

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

As a principal ingredient of vaccines, adjuvants can directly induce or enhance the powerful, widespread, innate, and adaptive immune responses associated ...

in-vitro-cellular-activity-evaluation-nanoemulsion-vaccine-adjuvant

Research

JoVE Journal

Immunology and Infection

1.7K Views.  Third Military Medical University of Chinese PLA. The protocol presents detailed methods for evaluating whether the nanoemulsion ophiopogonin D adjuvant promotes effective cellular immune responses. 

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

Human T Lymphocyte Isolation, Culture and Analysis of Migration In Vitro

The migration of T lymphocytes involves the adhesive interaction of cell surface integrins with ligands expressed on other cells or with extracellular matrix proteins. The precise spatiotemporal activation of integrins from a low affinity state to a high affinity state at the cell leading edge is important for T lymphocyte migration 1. Likewise, retraction of the cell trailing edge, or uropod, is a necessary step in maintaining persistent integrin-dependent T lymphocyte motility 2. Many therapeutic approaches to autoimmune or inflammatory diseases target integrins as a means to inhibit the excessive recruitment and migration of leukocytes 3. To study the molecular events that regulate human T lymphocyte migration, we have utilized an in vitro system to analyze cell migration on a two-dimensional substrate that mimics the environment that a T lymphocyte encounters during recruitment from the vasculature. T lymphocytes are first isolated from human donors and are then stimulated and cultured for seven to ten days. During the assay, T lymphocytes are allowed to adhere and migrate on a substrate coated with intercellular adhesion molecule-1 (ICAM-1), a ligand for integrin LFA-1, and stromal cell-derived factor-1 (SDF-1). Our data show that T lymphocytes exhibit a migratory velocity of ~15 &mu;m/min. T lymphocyte migration can be inhibited by integrin blockade 1 or by inhibitors of the cellular actomyosin machinery that regulates cell migration 2.

Human T Lymphocyte Isolation, Culture and Analysis of Migration In Vitro

T lymphocyte migration occurs during homing to lymphoid organs, exit from the vasculature, and entering into peripheral tissues. Here, we describe a protocol that can be used to analyze T lymphocyte migration in vitro.

The migration of T lymphocytes involves the adhesive interaction of cell surface integrins with ligands expressed on other cells or with extracellular ...

Quantitative Analyses of all Influenza Type A Viral Hemagglutinins and Neuraminidases using Universal Antibodies in Simple Slot Blot Assays

Hemagglutinin (HA) and neuraminidase (NA) are two surface proteins of influenza viruses which are known to play important roles in the viral life cycle and the induction of protective immune responses1,2. As the main target for neutralizing antibodies, HA is currently used as the influenza vaccine potency marker and is measured by single radial immunodiffusion (SRID)3. However, the dependence of SRID on the availability of the corresponding subtype-specific antisera causes a minimum of 2-3 months delay for the release of every new vaccine. Moreover, despite evidence that NA also induces protective immunity4, the amount of NA in influenza vaccines is not yet standardized due to a lack of appropriate reagents or analytical method5. Thus, simple alternative methods capable of quantifying HA and NA antigens are desirable for rapid release and better quality control of influenza vaccines.
Universally conserved regions in all available influenza A HA and NA sequences were identified by bioinformatics analyses6-7. One sequence (designated as Uni-1) was identified in the only universally conserved epitope of HA, the fusion peptide6, while two conserved sequences were identified in neuraminidases, one close to the enzymatic active site (designated as HCA-2) and the other close to the N-terminus (designated as HCA-3)7. Peptides with these amino acid sequences were synthesized and used to immunize rabbits for the production of antibodies. The antibody against the Uni-1 epitope of HA was able to bind to 13 subtypes of influenza A HA (H1-H13) while the antibodies against the HCA-2 and HCA-3 regions of NA were capable of binding all 9 NA subtypes. All antibodies showed remarkable specificity against the viral sequences as evidenced by the observation that no cross-reactivity to allantoic proteins was detected. These universal antibodies were then used to develop slot blot assays to quantify HA and NA in influenza A vaccines without the need for specific antisera7,8. Vaccine samples were applied onto a PVDF membrane using a slot blot apparatus along with reference standards diluted to various concentrations. For the detection of HA, samples and standard were first diluted in Tris-buffered saline (TBS) containing 4M urea while for the measurement of NA they were diluted in TBS containing 0.01% Zwittergent as these conditions significantly improved the detection sensitivity. Following the detection of the HA and NA antigens by immunoblotting with their respective universal antibodies, signal intensities were quantified by densitometry. Amounts of HA and NA in the vaccines were then calculated using a standard curve established with the signal intensities of the various concentrations of the references used. 
Given that these antibodies bind to universal epitopes in HA or NA, interested investigators could use them as research tools in immunoassays other than the slot blot only.

A simple slot blot method was developed for the quantification of influenza viral hemagglutinin and neuraminidase using universal antibodies targeting their most conserved sequences identified through bioinformatics analyses. This innovative approach may provide a useful alternative to quantitative determination of all viral hemagglutinin and neuraminidase.

Hemagglutinin (HA) and neuraminidase (NA) are two surface proteins of influenza viruses which are known to play important roles in the viral life cycle ...

Generation of Recombinant Arenavirus for Vaccine Development in FDA-Approved Vero Cells

The development and implementation of arenavirus reverse genetics represents a significant breakthrough in the arenavirus field 4. The use of cell-based arenavirus minigenome systems together with the ability to generate recombinant infectious arenaviruses with predetermined mutations in their genomes has facilitated the investigation of the contribution of viral determinants to the different steps of the arenavirus life cycle, as well as virus-host interactions and mechanisms of arenavirus pathogenesis 1, 3, 11 . In addition, the development of trisegmented arenaviruses has permitted the use of the arenavirus genome to express additional foreign genes of interest, thus opening the possibility of arenavirus-based vaccine vector applications 5 . Likewise, the development of single-cycle infectious arenaviruses capable of expressing reporter genes provides a new experimental tool to improve the safety of research involving highly pathogenic human arenaviruses 16 . The generation of recombinant arenaviruses using plasmid-based reverse genetics techniques has so far relied on the use of rodent cell lines 7,19 , which poses some barriers for the development of Food and Drug Administration (FDA)-licensed vaccine or vaccine vectors. To overcome this obstacle, we describe here the efficient generation of recombinant arenaviruses in FDA-approved Vero cells.

Rescue of recombinant arenaviruses from cloned cDNAs, an approach referred to as reverse genetics, allows researchers to investigate the role of specific viral gene products, as well as the contribution of their different specific domains and residues, to many different aspects of the biology of arenavirus. Likewise, reverse genetics techniques in FDA-approved cell lines (Vero) for vaccine development provides novel possibilities for the generation of effective and safe vaccines to combat human pathogenic arenaviruses.

The development and implementation of arenavirus reverse genetics represents a significant breakthrough in the arenavirus field  4. The use of cell-based ...

Immunostimulatory Agent Evaluation: Lymphoid Tissue Extraction and Injection Route-Dependent Dendritic Cell Activation

For evaluation of a new therapeutic agent for immunotherapy or vaccination, analysis of immune cell activation in lymphatic tissues is essential. Here, we investigated immunological effects of a novel lipid-DNA immunostimulant in nanoparticle form from different administration routes in the mouse: oral, intranasal, subcutaneous, footpad, intraperitoneal, and intravenous. These injections will directly influence the immune response, and harvesting lymphatic tissues and analysis of dendritic cell (DC) activation in the tissues are crucial parts of these evaluations. The extraction of mediastinal lymph nodes (mLNs) is important but quite complex because of the size and location of this organ. A stepwise procedure for harvesting the inguinal lymph node (iLN), mLN, and spleen and analyzing DC activation by flow cytometry is described.

Experimental procedures for the subsequent extraction of lymphatic tissues to test lymphoid dendritic cell activation are described after treatment of an immunostimulating nanomaterial.

For evaluation of a new therapeutic agent for immunotherapy or vaccination, analysis of immune cell activation in lymphatic tissues is essential. Here, we ...

Synthesis and Characterization of mRNA-Loaded Poly(Beta Aminoesters) Nanoparticles for Vaccination Purposes

Vaccination has been one of the major successes of modern society and is indispensable in controlling and preventing disease. Traditional vaccines were composed of entire or fractions of the infectious agent. However, challenges remain, and new vaccine technologies are mandatory. In this context, the use of mRNA for immunizing purposes has shown an enhanced performance, as demonstrated by the speedy approval of two mRNA vaccines preventing SARS-CoV-2 infection. Beyond success in preventing viral infections, mRNA vaccines can also be used for therapeutic cancer applications.
Nevertheless, the instability of mRNA and its fast clearance from the body due to the presence of nucleases makes its naked delivery not possible. In this context, nanomedicines, and specifically polymeric nanoparticles, are critical mRNA delivery systems. Thus, the aim of this article is to describe the protocol for the formulation and test of an mRNA vaccine candidate based on the proprietary polymeric nanoparticles. The synthesis and chemical characterization of the poly(beta aminoesters) polymers used, their complexation with mRNA to form nanoparticles, and their lyophilization methodology will be discussed here. This is a crucial step for decreasing storage and distribution costs. Finally, the required tests to demonstrate their capacity to in vitro transfect and mature model dendritic cells will be indicated. This protocol will benefit the scientific community working on vaccination because of its high versatility that enables these vaccines to prevent or cure a wide variety of diseases.

Synthesis and Characterization of mRNA-Loaded PolyBeta Aminoesters Nanoparticles for Vaccination Purposes

Here, a simple protocol is presented for producing mRNA nanoparticles based on poly(beta aminoester) polymers, easy to be tailored by changing the encapsulated mRNA. The workflow for synthesizing the polymers, the nanoparticles, and their in vitro essential characterization are also described. A proof-of-concept regarding immunization is also added.

Vaccination has been one of the major successes of modern society and is indispensable in controlling and preventing disease. Traditional vaccines were ...

Bioengineering

Preparation, Characteristics, Toxicity, and Efficacy Evaluation of the Nasal Self-Assembled Nanoemulsion Tumor Vaccine In Vitro and In Vivo

Epitope peptides have attracted widespread attention in the field of tumor vaccines because of their safety, high specificity, and convenient production; in particular, some MHC I-restricted epitopes can induce effective cytotoxic T lymphocyte activity to clear tumor cells. Additionally, nasal administration is an effective and safe delivery technique for tumor vaccines due to its convenience and improved patient compliance. However, epitope peptides are unsuitable for nasal delivery because of their poor immunogenicity and lack of delivery efficiency. Nanoemulsions (NEs) are thermodynamically stable systems that can be loaded with antigens and delivered directly to the nasal mucosal surface. Ile-Lys-Val-Ala-Val (IKVAV) is the core pentapeptide of laminin, an integrin-binding peptide expressed by human respiratory epithelial cells. In this study, an intranasal self-assembled epitope peptide NE tumor vaccine containing the synthetic peptide IKVAV-OVA257-264 (I-OVA) was prepared by a low-energy emulsification method. The combination of IKVAV and OVA257-264 can enhance antigen uptake by nasal mucosal epithelial cells. Here, we establish a protocol to study the physicochemical characteristics by transmission electron microscopy (TEM), atomic force microscopy (AFM), and dynamic light scattering (DLS); stability in the presence of mucin protein; toxicity by examining the cell viability of BEAS-2B cells and the nasal and lung tissues of C57BL/6 mice; cellular uptake by confocal laser scanning microscopy (CLSM); release profiles by imaging small animals in vivo; and the protective and therapeutic effect of the vaccine by using an E.G7 tumor-bearing model. We anticipate that the protocol will provide technical and theoretical clues for the future development of novel T cell epitope peptide mucosal vaccines.

Preparation, Characteristics, Toxicity, and Efficacy Evaluation of the Nasal Self-Assembled Nanoemulsion Tumor Vaccine In Vitro and In Vivo

Here, we present detailed methods for the preparation and evaluation of the nasal self-assembled nanoemulsion tumor vaccine in vitro and in vivo.

Epitope peptides have attracted widespread attention in the field of tumor vaccines because of their safety, high specificity, and convenient production; ...

Evaluating the Immune Response of a Nanoemulsion Adjuvant Vaccine Against Methicillin-Resistant Staphylococcus aureus (MRSA) Infection

Nanoemulsion adjuvant vaccines have attracted extensive attention because of their small particle size, high thermal stability, and ability to induce validly immune responses. However, establishing a series of comprehensive protocols to evaluate the immune response of a novel nanoemulsion adjuvant vaccine is vital. Therefore, this article features a rigorous procedure to determine the physicochemical characteristics of a vaccine (by transmission electron microscopy [TEM], atomic force microscopy [AFM], and dynamic light scattering [DLS]), the stability of the vaccine antigen and system (by a high-speed centrifuge test, a thermodynamic stability test, SDS-PAGE, and western blot), and the specific immune response (IgG1, IgG2a, and IgG2b). Using this approach, researchers can evaluate accurately&#160;the protective effect of a novel nanoemulsion adjuvant vaccine in a lethal MRSA252 mouse model. With these protocols, the most promising nanoemulsion vaccine adjuvant in terms of effective adjuvant potential can be identified. In addition, the methods can help optimize novel vaccines for future development.

Evaluating the Immune Response of a Nanoemulsion Adjuvant Vaccine Against Methicillin-Resistant Staphylococcus aureus MRSA Infection

The present protocol prepares and evaluates the physical properties, immune response, and in vivo protective effect of a novel nanoemulsion adjuvant vaccine.

Nanoemulsion adjuvant vaccines have attracted extensive attention because of their small particle size, high thermal stability, and ability to induce ...

Evaluating Immunogenicity of Nanoemulsion&#45;Encapsulated Retinoic Acid in Mice

Cationic Nanoemulsion-Encapsulated Retinoic Acid as an Adjuvant to Promote OVA&#45;Specific Systemic and Mucosal Responses

Cationic nanostructures have emerged as an adjuvant and antigen delivery system that enhances dendritic cell maturation, ROS generation, and antigen uptake and then promotes antigen-specific immune responses. In recent years, retinoic acid (RA) has received increasing attention due to its effect in activating the mucosal immune response; however, in order to use RA as a mucosal adjuvant, it is necessary to solve the problem of its dissolution, loading, and delivery. Here, we describe a cationic nanoemulsion-encapsulated retinoic acid (CNE-RA) delivery system composed of the cationic lipid 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOTAP), retinoic acid, squalene as the oil phase, polysorbate 80 as surfactant, and sorbitan trioleate 85 as co-surfactant. Its physical and chemical properties were characterized using dynamic light scattering and a spectrophotometer. Immunization of mice with the mixture of antigen (ovalbumin, OVA) and CNE-RA significantly elevated the levels of anti-OVA secretory immunoglobulin A (sIgA) in vaginal lavage fluid and the small intestinal lavage fluid of mice compared with OVA alone. This protocol describes a detailed method for the preparation, characterization, and evaluation of the adjuvant effect of CNE-RA.

Author Spotlight: Development and Evaluation of a Cationic Nanoemulsion&#45;Encapsulated Retinoic Acid System for Mucosal Vaccination

In this protocol, we developed a cationic nanoemulsion-encapsulated retinoic acid (RA) to be used as an adjuvant to promote antigen-specific systemic and mucosal responses. By adding the FDA-approved RA to the nanoemulsion, antigen-specific sIgA was promoted in the vagina and small intestine after intramuscular injection of the nanoemulsion.

Cationic nanostructures have emerged as an adjuvant and antigen delivery system that enhances dendritic cell maturation, ROS generation, and antigen ...

Preparation of a Nanoemulsion Adjuvant Vaccine

Source: Zhang, Z. et al., <a href="https://app.jove.com/v/64299">Preparation, Characteristics, Toxicity, and Efficacy Evaluation of the Nasal Self-Assembled Nanoemulsion Tumor Vaccine In Vitro and In Vivo.</a>&#160;J. Vis. Exp. (2022)
This video demonstrates the production of nanoemulsion tumor vaccines. The stable oil reservoir depots carrying synthetic peptides and adjuvants are produced by low-energy nanoemulsion, offering significant promise for tumor immunotherapy.

1. Preparation of the I-OVA NE

	Admix 1 mg of monophosphoryl lipid A (MPLA) with 100 &#181;L of dimethyl sulfoxide (DMSO), vortex for 5 min, and allow it ...

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Immunology

Immunotherapy

Immunogenics and Vaccine Development

Measuring Immunoglobulin G Levels Against a Test Vaccine in a Mouse Serum

Source: Zeng, X. et al., <a href="https://app.jove.com/v/65152">Evaluating the Immune Response of a Nanoemulsion Adjuvant Vaccine Against Methicillin-Resistant Staphylococcus aureus (MRSA) Infection.</a>&#160;J. Vis. Exp.&#160;(2023)
This video demonstrates a method for assessing the antibody immune response to a novel nanoemulsion adjuvant vaccine in pre-immunized mice. Immunization enhances antibody response as confirmed by determining the IgG antibody titers.

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
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In Vitro Cellular Activity Evaluation of the Nanoemulsion Vaccine Adjuvant Ophiopogonin D

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Synthesis and Characterization of mRNA-Loaded Poly(Beta Aminoesters) Nanoparticles for Vaccination Purposes

Preparation, Characteristics, Toxicity, and Efficacy Evaluation of the Nasal Self-Assembled Nanoemulsion Tumor Vaccine In Vitro and In Vivo

Evaluating the Immune Response of a Nanoemulsion Adjuvant Vaccine Against Methicillin-Resistant Staphylococcus aureus (MRSA) Infection

Cationic Nanoemulsion-Encapsulated Retinoic Acid as an Adjuvant to Promote OVA-Specific Systemic and Mucosal Responses

Preparation of a Nanoemulsion Adjuvant Vaccine

Measuring Immunoglobulin G Levels Against a Test Vaccine in a Mouse Serum