Cd86 apc

Manufactured by Thermo Fisher Scientific

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The CD86-APC is a fluorescently labeled monoclonal antibody that binds to the CD86 cell surface protein. CD86 is a costimulatory molecule expressed on antigen-presenting cells and plays a role in the activation of T cells. The APC fluorescent label allows for the detection and analysis of CD86-expressing cells using flow cytometry techniques.

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CD86 (B7-2)-APC APC)-conjugated anti-mouse CD86 APC-labeled anti-CD86 Anti-mouse CD86-APC APC-conjugated anti-CD86 (GL-1) Anti-CD86-APC (clone GL1; Anti-CD86-APC

32 protocols using cd86 apc

Plasmid-based PTEN Gene Therapy

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The three plasmid systems, including target gene plasmid (pAAV-CAG-mCherry), capsid plasmid (pAAV-6, pAAV-1, pAAV-7m8, pAAV-7, pAAV-8, pAAV-10) and helper plasmid (pHelper), were all purchased from Fenghui Biotechnology (Hunan, China). The complementary DNA (cDNA) encoding the mouse PTEN gene was synthesized from GENEWIZ (Suzhou, China). pAAV-CAG-PTEN was obtained by replacing mCherry gene in pAAV-CAG-mCherry with PTEN gene. Reverse the PTEN gene sequence in pAAV-CAG-PTEN to get pAAV-CAG-PTEN^R.
The anti-PD-1 used in this study is AUNP-12, a polypeptide purchased from Selleck (USA) with a purity of 99.20%. Cy5-AUNP-12 was purchased from Guoping pharmaceutical co (Anhui, China) with a purity of 90%. CpG was synthesized in GENEWIZ (Suzhou, China) with 90% purity.
Antibodies for Western blot (WB) and Immunofluorescence (IF): anti-mouse PTEN, CRT, β-actin and anti-rabbit FITC, CY3 fluorescent secondary antibodies were purchased from ABclonal Tech (Wuhan, China). Antibodies for flow cytometry test: CD11c-PE, CD80-FITC, CD86-APC, CD3-APC, CD8-FITC, CD4-APC-eflour780, CD69-PE, CD274-PE, CD11b-FITC, CD206-PE-Cy7, CD86-APC, IL-4-PE, IFN-γ-APC, TNF-α-eflour450, IL-2-PE5.5, CD4-APC-eflour780, CD44-APC, CD62L-PE were all purchased from Invitrogen (USA).

Zhang Y., Yang L., Ou Y., Hu R., Du G., Luo S., Wu F., Wang H., Xie Z., Zhang Y., He C., Ma C., Gong T., Zhang L., Zhang Z, & Sun X. (2023). Combination of AAV-delivered tumor suppressor PTEN with anti-PD-1 loaded depot gel for enhanced antitumor immunity. Acta Pharmaceutica Sinica. B, 14(1), 350-364.

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Disulfiram and Copper Gluconate Protocol

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Disulfiram (Selleck, S1680) and copper gluconate (CuGlu, Sangon Biotech, A503014) with over 99% purity were used for the study. The antibody to NPL4 (sc-365796) was obtained from Santa Cruz. The antibodies to EIF2S1 (ab32157), XBP1 (ab220783), and EIF2S1 (phosphor S51) (ab32157) were obtained from Abcam. The antibodies to CHOP (A0221), Ubiquitin (A18185), β-tubulin (A7074), and Lamin-B (A19970) were obtained from Abclonal. The antibodies to Zombie NIR™ APC-CY7(423106), CD45 FITC (103108)/PE (103106), CD11c PE (117308)/APC (117310), I-Ab PE-CY7 (116420), CD80 PE-CY5.5 (104712), CD86 APC (105012), CD3 APC (100236), CD8 PE (100708)/APC-CY7 (100712), CD4 PE (130310), CD44 PE-CY7(103010), CD62L APC (104412), Granzyme B PE-CY7(372213), and IFN-γ PE-CY5.5(505821) were obtained from eBioscience (San Diego, CA, USA).

Gao X., Huang H., Pan C., Mei Z., Yin S., Zhou L, & Zheng S. (2022). Disulfiram/Copper Induces Immunogenic Cell Death and Enhances CD47 Blockade in Hepatocellular Carcinoma. Cancers, 14(19), 4715.

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Comprehensive Immune Cell Profiling by Flow Cytometry

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Flow cytometry was performed as previously described [38 (link)]. Briefly, cells were incubated with LIVE/DEAD Fixable cell stain (Invitrogen), then stained for 20 min with: MHCII-PacificBlue, F4/80-APC (Biolegend), CD4-AF700, CD8-PacificBlue, CD25-APC, CD86-APC, CD49b-Pe-Cy7 (eBioscience), CD11b-PE-Cy7, CD45R/B220-PerCP-Cy5.5 (BD-Pharmingen). For intracellular staining, the samples were stimulated for 3 h (100 ng/ml phorbol 12-myristate 13-acetate, 1 μg/ml ionomycin; monensin added after 1 h). Cells were fixed and permeabilized with Cytofix/Cytoperm (BD), then stained with IL-10-FITC, IFN-γ-PerCP-Cy5.5, IL-17-PE, FoxP3-AF488 (BD-Pharmingen) antibodies before analysis on LSRII flow cytometer (BD), collecting 100,000 live events. Data was analyzed using FlowJo (v7.6.5, Tree Star, Ashland, OR, USA).

Schuijs M.J., Hartmann S., Selkirk M.E., Roberts L.B., Openshaw P.J, & Schnoeller C. (2016). The Helminth-Derived Immunomodulator AvCystatin Reduces Virus Enhanced Inflammation by Induction of Regulatory IL-10+ T Cells. PLoS ONE, 11(8), e0161885.

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Monoclonal Antibodies for CHIKV Study

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The mouse anti-CHIKV-nsP2 monoclonal antibody, used in this study was developed by us [58 (link)]. The anti-CHIKV-E2 monoclonal antibody was a kind gift from Dr. Manmohan Parida, DRDE, Gwalior, India. HRP linked secondary antibodies, H-2k^d PE, I-A/I-E PE, isotype PE, isotype APC and HSP90 antibodies were purchased from BD Biosciences (San Jose, CA, USA). CD86 APC and CD80 APC were purchased from eBiosciences (San Diego, CA, USA). The monoclocal antibodies for cleaved caspase-3 (Asp175), cleaved caspase-8 (Asp387) and caspase-9 (C9) were purchased from cell signaling technology (Danvers, MA, USA). The anti-mouse Alexa Fluor 488 was purchased from Invitrogen (Carlsbad, CA, USA). Mouse IgG1 isotype control, GAPDH and β-actin were purchased from Abgenex India Pvt. Ltd. (Bhubaneswar, India). Saponin, Anisomycin and Bovine serum albumin fraction V were purchased from Sigma-Aldrich. 17-Allylaminogeldanamycin (17-AAG) and Z-VAD-FMK were purchased from Merck Millipore (Billerica, MA, USA).

Nayak T.K., Mamidi P., Kumar A., Singh L.P., Sahoo S.S., Chattopadhyay S, & Chattopadhyay S. (2017). Regulation of Viral Replication, Apoptosis and Pro-Inflammatory Responses by 17-AAG during Chikungunya Virus Infection in Macrophages. Viruses, 9(1), 3.

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Multiparameter Flow Cytometry Immunophenotyping

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Fc-gamma receptors were blocked with a rat anti-mouse CD16/CD32 antibody (BD). Mouse-specific antibodies were the following: CD3-Brilliant violet 510, CD25-PercP Cy5.5, I-A/I-E-Brilliant violet 510, CD45R/B220-PE-Cy7, CD43-APC, CTLA-4-APC (all from BioLegend), CD4-APC-Cy7, CD69-PE, CD11c-PercP Cy5.5, CD19-APC-Cy7, CD40-FITC (all from BD Pharmingen), CD8α-PE-Cy7, FoxP3-FITC, CD86-APC, CD83-FITC, CD80-PE-Cy7, IgM-PercP-eFluor 710 (all from eBioscience).
During the experimental set-up, CD3 antibody was included in the staining panel (BV510 BioLegend clone 17A2) and used for the gating of T cells. The results obtained with gating on CD3+ CD4+ CD8- were similar to those obtained with gating on CD4+ CD8-. Due to the limitation in the maximum number of colours that can be discriminated with the FACS Canto II, CD3 staining was omitted in subsequent stainings.
Dead cells were excluded using the fixable viability dye-eFluor 450 (eBioscience), and intracellular staining was performed using the fixation/ permeabilization buffer set from eBioscience. Flow cytometry measurements were performed on a FACS Canto II instrument (BD) and the data were analyzed with FlowJo software (Tree Star).

Sordé L., Spindeldreher S., Palmer E, & Karle A. (2017). Massive immune response against IVIg interferes with response against other antigens in mice: A new mode of action?. PLoS ONE, 12(10), e0186046.

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Dendritic Cell Activation Assay

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PRT, OVA, propidium iodide (PI) and lipopolysaccharides (LPS) were purchased from Sigma-Aldrich (St. Louis, MO, USA). CpG 1826 (5′-TCC ATG ACG TTC CTG ACG TT-3′) was bought from Sangon (Shanghai, China). Granulocyte-macrophage colony stimulating factor (GM-CSF) and interleukin-4 (IL-4) from mouse were bought from Novus Biologicals (Colorado, USA). The antibodies such as CD11c-PE, CD86-APC, CD80-FITC, CD3e-PE, CD8a-FITC and OVA_257-264 (SIINFEKL) peptide bound to H-2 K^b APC for flow cytometry (FCM) detection, and the biotinylated OVA_257-264 (SIINFEKL) peptide bound to H-2 K^b monoclonal antibody for immunofluorescence, these antibodies were all bought from eBioscience (CA, USA). The ELISA kits for mouse interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ) were bought from R&D Systems (Minneapolis, MA, USA). The aPD-1 (InVivoMab anti-mouse PD-1) was purchased from BioXcell (New Hampshire, USA). Avidin-FITC was bought from Boster Bio-Tech (Wuhan, China).

Jiang M., Zhao L., Cui X., Wu X., Zhang Y., Guan X., Ma J, & Zhang W. (2021). Cooperating minimalist nanovaccine with PD-1 blockade for effective and feasible cancer immunotherapy. Journal of Advanced Research, 35, 49-60.

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In Vivo Uptake of Tumor Cells

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C57BL/6 female mice (6–9 weeks old) were injected intra-peritoneum with PBS containing 3 × 10⁶ B16-F10 cells (BNIP3^WT or BNIP3^KD) pHrodo-labelled (Life Technologies, P36600). The peritoneal cells were collected 24 h post-injection via peritoneal lavage with Ca²⁺- and Mg²⁺-free PBS. The cells were transferred to an ultra-low attachment V-bottom plate and stained with the fixable Live/Dead Yellow stain (Invitrogen). After blocking the F_c receptor (CD16/32, eBioscience, 16-0161-82), cells were stained for F4/80-eFluor780 (eBioscience, 47-4801-80), CD11b-eFluor450 (eBioscience, 48-0112-82), MHCII-FITC (eBioscience, 11-5321-81), CD86-APC (eBioscience, 17-0862-81), CD206-PeCy7 (eBioscience, 25-2061-80) in FC buffer. Samples were acquired on a Gallios^™ flow cytometer and the data analysis was performed via FlowJo_V10^™ software.

Romano E., Rufo N., Korf H., Mathieu C., Garg A.D, & Agostinis P. (2018). BNIP3 modulates the interface between B16-F10 melanoma cells and immune cells. Oncotarget, 9(25), 17631-17644.

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Characterizing Microbiome-Host Interactions

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EcN and Salmonella typhimurium were purchased from China General Microbiological Culture Collection Center. Caco-2 and Raji cell lines were obtained from the American Type Culture Collection. Baker’s yeast was obtained from Lesaffre. Minimum Eagle’s medium (MEM), RPMI 1640, fetal bovine serum, and antibiotic/antimycotic solution were purchased from Thermo Fisher Scientific. Calcofluor-white stain was purchased from Sigma-Aldrich. The β-Glucan assay kit was purchased from Megazyme. Fluorescence-activated cell sorting antibodies including anti-mouse CD3-PerCP Cy5.5, CD4–fluorescein isothiocyanate (FITC), CD8–allophycocyanin (APC), CD11c-FITC, CD80–phycoerythrin (PE), CD86-APC, CD45R/B220-PE-Cy7, and CD138-APC were purchased from eBioscience. IgA-PE was purchased from Thermo Fisher Scientific. Plasmids pBBR1MCS2-Tac-mCherry (kanamycin resistance) and all other reagents were purchased from domestic suppliers and used as received.

Lin S., Mukherjee S., Li J., Hou W., Pan C, & Liu J. (2021). Mucosal immunity–mediated modulation of the gut microbiome by oral delivery of probiotics into Peyer’s patches. Science Advances, 7(20), eabf0677.

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Cytokine and Surface Marker Analysis of Bone Marrow-Derived Dendritic Cells

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BMDCs were cultured with different stimuli for 48 h. Supernatants were collected and assayed for cytokine production using the CBA Mouse Inflammation Kit (IL-6, IL-10, IFN-γ, TNF-α, and IL-12p70) (BD Biosciences, Cat. # 552364), according to the manufacturer’s instructions. Cells were stained with: CD11c-PE (eBioscience, Cat. #12-0114, dilution 1/50), CD86-APC (eBioscience, Cat. # 17-0862, dilution 1/250) and CD40-PerCP-eFluo710 (eBioscience, Cat. # 46-0401, dilution 1/50) and analyzed in an Accuri® C6 flow cytometer (BD). Gating strategy is shown in Supplementary Fig. 3a. Appropriate isotype controls were used.

Serradell M.C., Rupil L.L., Martino R.A., Prucca C.G., Carranza P.G., Saura A., Fernández E.A., Gargantini P.R., Tenaglia A.H., Petiti J.P., Tonelli R.R., Reinoso-Vizcaino N., Echenique J., Berod L., Piaggio E., Bellier B., Sparwasser T., Klatzmann D, & Luján H.D. (2019). Efficient oral vaccination by bioengineering virus-like particles with protozoan surface proteins. Nature Communications, 10, 361.

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Assessing BMDC Activation and Viability

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To assess BMDC activation status and viability after CS treatment, BMDCs were treated for 24 h. After treatment, cells were harvested and stained for CD11b BV421 (BioLegend, clone: M1/70), CD11c APC Fire 750 (BioLegend, clone: N418), I‐A/I‐E major histocompatibility complex (MHC) Class II V500 (BD Biosciences, clone: M5/114.15.2), CD40 PE‐Cy7 (BioLegend, clone: 3/23), CD80 PE (BD Pharmingen, clone: 16‐10‐A1), CD86 APC (eBiosciences, clone: GL1), Annexin V PE (BD Biosciences), and 7‐aminoactinomycin D (7AAD; BD Biosciences). Samples were analyzed on a Cytek DxP10 (Cytek Biosciences, Inc.) flow at the University of Nebraska‐Lincoln Flow Cytometry Service Center. Data were analyzed using FlowJo software (Becton, Dickinson and Company).

Lampe A.T., Farris E.J., Brown D.M, & Pannier A.K. (2020). High‐ and low‐molecular‐weight chitosan act as adjuvants during single‐dose influenza A virus protein vaccination through distinct mechanisms. Biotechnology and Bioengineering, 118(3), 1224-1243.

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