DCs from patients with T1D (n = 7) were co-cultured with 1 mM PSAB-liposomes (PSAB-DC), 1000 nM Lira (Lira-DC) or combined (PSAB + Lira DC) for 24 h in the presence of 20 μg/ml human insulin (Sigma-Aldrich). DCs were cultured with 20 μg/ml human insulin (Sigma-Aldrich) to obtain immature DCs (iDCs) and adding a cytokine cocktail [1000 IU/ml TNFα and 2000 IU/ml IL-1β (Immunotools) and 1 μM Prostaglandin E2 (Cayman Chemical, Ann Arbor, USA)] to obtain mature DCs (mDC). To assess DCs phenotype, CD25-PE, CD86-FITC, HLA ABC-FITC, HLA DR-FITC, CD14-PE and CD40-APC (Immunotools), CD36-APCCy7, TIM4-APC, CD54-PECy7, TLR2-FITC, CXCR4-APCCy7, CCR2-APC, PD-L1-PECy7, ILT3-PECy7 (Biolegend, San Diego, USA) and CCR7-PECy7 (BD Biosciences) monoclonal antibodies were used to determine their membrane expression. All DCs conditions from the same patient were analysed at the time. All MFI groups were normalized to the MFI of mDCs.
Cd86 fitc
Manufactured by Immunotools
Sourced in United States
CD86-FITC is a fluorescently-labeled antibody that binds to the CD86 cell surface protein. CD86 is a co-stimulatory molecule expressed on antigen-presenting cells, such as dendritic cells and B cells, and plays a role in T cell activation.
Automatically generated - may contain errors
Lab products found in correlation
Human insulin, by Merck Group (2 mentions)
Hla dr fitc, by Immunotools (2 mentions)
Cd40 apc, by Immunotools (2 mentions)
Cd36 apccy7, by Immunotools (2 mentions)
Tim4 apc, by BioLegend (2 mentions)
Cd54 pecy7, by BioLegend (2 mentions)
Tlr2 fitc, by BioLegend (2 mentions)
Cxcr4 apccy7, by BioLegend (2 mentions)
Ccr2 apc, by BioLegend (2 mentions)
Prostaglandin e2, by Cayman Chemical (2 mentions)
Cd14 pe, by Immunotools (2 mentions)
Il 1β, by Immunotools (2 mentions)
Hla abc fitc, by Immunotools (2 mentions)
7 aad, by BD (2 mentions)
Ccr7 pe cy7, by BD (1 mentions)
Pd l1 pe cy7, by BioLegend (1 mentions)
Facscanto 2, by BD (1 mentions)
Facscalibur flow cytometer, by BD (1 mentions)
Facscanto 2 flow cytometer, by BD (1 mentions)
Diva software, by BD (1 mentions)
4 protocols using cd86 fitc
1
Immunomodulatory Effects of PSAB-liposomes and Liraglutide on Dendritic Cells in Type 1 Diabetes
2
Characterizing Tolerogenic Dendritic Cells
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DCs from patients at onset and with established disease were cultured for 24 h with 20 μg/mL human insulin (Sigma-Aldrich, St. Louis, MO, USA) and 1 mmol/L of PSAB-liposomes to determine the effect of insulin chains as autoantigens (tolDCs). As controls, DCs were either cultured with 20 μg/mL human insulin (Sigma-Aldrich) to obtain iDCs or adding a cytokine cocktail —TNF-α (1,000 IU/mL, Immunotools), IL-1β (2,000 IU/mL, Immunotools), and Prostaglandin E2 (PGE2, 1 μmol/L, Cayman Chemical, Ann Arbor, MI, USA)— for 24 h to obtain mature DCs (mDCs). Viability and phenotype were analyzed by flow cytometry (FACSCanto II, BD Biosciences). DCs were stained with 7-AAD (BD Biosciences) and antibodies to CD11c-APC, CD86-FITC, HLA-ABC-FITC, HLA-DR-FITC, CD14-PE and CD40-APC (Immunotools), CD36-APCCy7, TIM4-APC, αvβ5 integrin-PE, CD54-PECy7, TLR2-FITC, CXCR4-APCCy7, CCR2-APC (BioLegend, San Diego, CA, USA). Data were analyzed using FlowJo software (Tree Star Inc, Ashland, OR, USA).
3
Maturation of Immature Dendritic Cells
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Immature DCs were treated with different amounts of NLC, diluted from formulations described in Table S1 , in presence or absence of 100 ng/mL of TNF-α (Immunotools), and allowed to mature for 72 hours, before being harvested. Flow cytometry was performed as previously described.25 (link) Briefly, cells were washed and labeled (for 45 minutes at 4°C in the dark) in staining buffer with combinations of the following antihuman antibodies: CD1a, CD11c, CD86-FITC, CD40-FITC, HLA-ABC-PE, human leukocyte antigen – antigen D related (HLA-DR-PE) (all from Immunotools), CD83-FITC (AbDSerotec, Kidlington, UK). Isotype and fluorochrome-matched control antibodies were used to define background staining. For cell viability, Annexin V-FITC staining was performed for 15 minutes, followed by propidium iodide (PI; BD Biosciences, San Jose, CA, USA) staining, just prior to acquisition. For each sample, 10,000 cells were acquired, gated according to forward and side scatter parameters. Samples were analyzed using a FACS Calibur flow cytometer with Cell Quest software, or FACS Canto II flow cytometer, with Diva software (all from BD Biosciences, Franklin Lakes, New Jersey, USA). Results were analyzed using FlowJo software (TreeStar, Inc., Ashland, OR, USA). The mean fluorescence intensity for each sample was calculated subtracting the mean fluorescence intensity of the respective isotype control.
4
Comprehensive Phenotyping of Immune Cells
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The cell phenotype was analyzed using the following antibodies from BD Bioscience: HLA-DR-FITC (Tü39 or L243), DC-SIGN/CD209-PerCP-Cy5.5 (DCN46), CD1a-APC (HI149), CD86-FITC (FUN-1), CD83-APC (HB15e), and CD14-PE (MEM-15, ImmunoTools). Data were acquired on a FACSCalibur (Becton-Dickinson) or Gallios (Beckman-Coulter) after exclusion of dead cells by Sytox Red (Molecular Probes, Invitrogen) or 7-AAD (BD-Pharmingen) DNA intercalating dyes. Data were analyzed using the Cell Quest Pro software (BD Bioscience) or FlowJo (Treestar).
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