The in vitro functional ability of IL-10 modRNA-translated proteins was examined in a T cell line (Jurkat, clone E6-1, BCRC-60424). Jurkat cells (3 × 105 cells per well) were transfected with 200 ng IL-10 modRNA and seeded in the top chambers of Corning HTS Transwell 96-well plates (Merck, Darmstadt, Germany). Mouse C57BL/6J splenocytes (3 × 105 cells per well) were activated with coated anti-mouse CD3 (2 μg/mL; BioLegend, San Diego, CA) and soluble anti-mouse CD28 (2 μg/mL; BioLegend) and seeded in the bottom reservoir. At different time points, the supernatants in the bottom reservoir were harvested and analyzed by mouse IL-2 and IFN-γ ELISA (R&D Systems) according to the manufacturer’s instructions. A SpectraMax reader was used to read the absorbance at 450 nm.
Soluble anti mouse cd28
Manufactured by BioLegend
Soluble anti-mouse CD28 is a laboratory reagent used in immunological research. It is an antibody that binds to the CD28 receptor found on the surface of mouse T cells. CD28 is a co-stimulatory molecule that plays a crucial role in T cell activation and proliferation. The soluble form of the anti-CD28 antibody can be used in experimental settings to study T cell biology and immune responses.
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Lab products found in correlation
Mog35 55, by Merck Group (2 mentions)
Anti mouse cd3, by BioLegend (2 mentions)
Countess automated cell counter, by Thermo Fisher Scientific (1 mentions)
Anti mouse cd3e, by BioLegend (1 mentions)
Vybrant fam caspase 3 and 7 assay kit, by Thermo Fisher Scientific (1 mentions)
Bio plex magpix multiplex reader, by Bio-Rad (1 mentions)
Glomax multi microplate multimode reader, by Promega (1 mentions)
Anti human cd3, by BioLegend (1 mentions)
Anti mouse cd3, by Thermo Fisher Scientific (1 mentions)
Soluble anti human cd28, by BioLegend (1 mentions)
Gentlemacs tissue dissociator, by Miltenyi Biotec (1 mentions)
Propidium iodide, by Merck Group (1 mentions)
Celltrace violet cell proliferation reagent, by Thermo Fisher Scientific (1 mentions)
Celltiter glo, by Promega (1 mentions)
Protein transport inhibitor, by Thermo Fisher Scientific (1 mentions)
7 protocols using soluble anti mouse cd28
1
Evaluating IL-10 modRNA Effects on T Cells
2
Ex Vivo Lymph Node Cell Proliferation Assay
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The mesenteric lymph nodes were isolated from naïve or infected mice and kept in sterile PBS. Harvested lymph nodes from individual mouse were passed through 70 μm cell strainer for single cell suspension. The cell viability was assessed using Trypan blue and counted using Countess automated cell counter (Invitrogen). The cells were resuspended in DMEM and adjusted to 2×105 cells per well. A solution of anti-mouse CD3e (100314, Biolegend) was prepared at 5 μg/mL in sterile PBS and 50 μl of antibody solution was added into 96 well plate followed by incubation at 37°C for 2 hours. The antibody solution was removed with a multichannel pipettor and single cell suspension of lymph node cells (2×105 cells per well) was added to respective wells in coated 96 well plates. Next, soluble anti-mouse CD28 (102112, Biolegend) was added to cells at 2 μg/mL. Cells were incubated for 4 days to induce proliferation of cells. After incubation, cells were centrifuged and supernatant was collected for cytokine analysis by ELISA.
3
Caspase-3/7 Activity and Cell Viability
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The Vybrant™ FAM Caspase-3 and -7 Assay Kit (Life Technologies, Thermo Fisher Scientific) was used to evaluate cell viability by simultaneously analyzing caspase activity and cell membrane permeability by flow cytometry. Naïve CD3+ enriched T-cells were isolated as described above and stimulated for 24 h with plate-bound anti-mouse CD3 (3 µg/ml, BioLegend) and soluble anti-mouse CD28 (3 µg/ml, BioLegend). Effector/memory T-cells were isolated from previously MOG35–55-immunized mice as described above and stimulated with MOG35–55 (50 µg/ml, Sigma-Aldrich). Cells were then stained following the manufacturer’s protocol. Briefly, 300 µl of cell suspension at a concentration of 106 cells/ml were incubated with FLICA 30× working solution during 1 h at 37°C in the dark. Subsequently, cells were washed and incubated on ice with 2 µl of PI for at least 5 min before flow cytometric analysis on the LSRII (BD Biosciences). Data analysis was done using the FlowJo V10 software (Tree Star).
4
Cytokine Profiling of RhoA Mutant T-cells
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Culture supernatant from RhoA+/+ and RhoA−/− CD3+ T-cells was collected after 24, 48, and 72 h of stimulation with plate-bound anti-mouse CD3 (3 µg/ml, BioLegend) and soluble anti-mouse CD28 (3 µg/ml, BioLegend) in case of naïve T-cells, or with MOG35–55 (50 µg/ml, Sigma-Aldrich) for effector/memory T-cells isolated from mice immunized with MOG35–55 11 days before. We used the Essential Th1/Th2 Cytokine 6-plex Mouse ProcartaPlex™ Panel (Invitrogen, Thermo Fisher Scientific), which targets the mouse cytokines IFN-γ, IL-12p70, IL-6, TNF-α, IL-4, and IL-5. For the determination of IL-17A, the Mouse IL-17A uncoated ELISA kit (Invitrogen) was used. Each sample was processed according to the manufacturer’s instructions. Samples were analyzed in duplicates, and cytokine concentrations were interpolated from the standard curve. The multiplex plate was read in the Bio-Plex® MAGPIX™ Multiplex reader (BioRad, Hercules, CA, USA) and data acquired using the Bio-Plex Manager Software v6.1. The ELISA plate was read in the GloMax®-Multi Microplate Multimode Reader (Promega, Madison, WI, USA).
5
Measuring MDSC-mediated T-cell Suppression
6
Ex Vivo T Cell Activation and Antigen Cross-Presentation Assays
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For ex vivo T cell activation experiments, mice were euthanized and spleens were removed and mechanically disrupted with a GentleMacs tissue dissociator (Miltenyi Biotec, Inc.). Total spleen cells were washed with sterile PBS, counted, and CD8+ or CD4+ T cells were magnetically separated with CD8+ or CD4+ separation kits (19853 and 19852, respectively; Stemcell Technologies).
For CD3/CD28-mediated activation, ultra low-endotoxin, azide-free plate-bound anti-mouse CD3 (100223; BioLegend) was used at 2 μg/ml, and soluble anti-mouse CD28 (102116; BioLegend) was used at 8 μg/ml. T cells were incubated for 72 h prior to flow cytometry analysis or Cell Titer Glo (G7570; Promega) analysis.
For antigen cross-presentation assays, 2 × 104 BMDCs were plated, activated with LPS (10 μg/ml) for 2 h, and pulsed with SIINFEKL (100 nM), ovalbumin, EF-OVA, or CT26 lysate (all at 250 μg/ml) overnight. BMDCs were then washed with media, and 2 × 105 CD8+ or CD4+ T cells pre-stained with Celltrace Violet Cell Proliferation reagent (C34557; Thermo Fisher Scientific) were added and co-incubated for 72 h. Proliferation was then determined by loss of Celltrace Violet signal in propidium iodide (Sigma-Aldrich)–negative, viable CD8+, or CD4+ T cells after co-incubation.
For CD3/CD28-mediated activation, ultra low-endotoxin, azide-free plate-bound anti-mouse CD3 (100223; BioLegend) was used at 2 μg/ml, and soluble anti-mouse CD28 (102116; BioLegend) was used at 8 μg/ml. T cells were incubated for 72 h prior to flow cytometry analysis or Cell Titer Glo (G7570; Promega) analysis.
For antigen cross-presentation assays, 2 × 104 BMDCs were plated, activated with LPS (10 μg/ml) for 2 h, and pulsed with SIINFEKL (100 nM), ovalbumin, EF-OVA, or CT26 lysate (all at 250 μg/ml) overnight. BMDCs were then washed with media, and 2 × 105 CD8+ or CD4+ T cells pre-stained with Celltrace Violet Cell Proliferation reagent (C34557; Thermo Fisher Scientific) were added and co-incubated for 72 h. Proliferation was then determined by loss of Celltrace Violet signal in propidium iodide (Sigma-Aldrich)–negative, viable CD8+, or CD4+ T cells after co-incubation.
7
Characterizing CD8+ T cell function
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