Cell surface antigens were used to characterize the MSC properties by flow cytometry. The cells were detached with 0.25% trypsin-EDTA and centrifuged at 3,700 rpm for 6 min at room temperature to obtain the cell pellets. Non-specific binding was then blocked with 10% human AB serum at 4 °C for 30 min. For the detection of Oct-4, the cells were permeablised with 1% Triton X-100 (amresco®, Ohio, USA) for 1 min before incubated with antibody. Detroit 551 (fibroblast) (Sigma-Aldrich, USA) was used a negative control for the detection of Oct-4. Subsequently, cells pellets were incubated with the following monoclonal antibodies: Mouse anti-human CD31-PE (Immuno Tools GmbH, Germany), CD34-FITC (Immuno Tools GmbH, Germany), CD44-PE (Pierce Biotechnology, USA), CD45-PE (Immuno Tools GmbH, Germany), CD73-PE (Immuno Tools GmbH, Germany), CD90-FITC (Biolegend, USA), CD105-PE (Pierce Biotechnology, USA), HLA-ABC-FITC (Immuno Tools GmbH, Germany), HLA-DR-PE (Immuno Tools GmbH, Germany), and Oct-4-Alexa Fluor® 488 conjugate (Sigma-Aldrich, USA). Isotype antibodies served as a control to exclude nonspecific binding. Quantitative analysis was performed using FACScan (BD Biosciences) and the results were analyzed using CellQuest™ Pro 9.0 software (BD Biosciences).
Hla abc fitc
Manufactured by Immunotools
Sourced in United States
HLA-ABC-FITC is a fluorescently labeled antibody that binds to the major histocompatibility complex (MHC) class I proteins, also known as human leukocyte antigen (HLA)-A, -B, and -C. This product is commonly used in flow cytometry applications to identify and quantify cells expressing these surface markers.
Automatically generated - may contain errors
Lab products found in correlation
Cd40 apc, by Immunotools (3 mentions)
Cellquest pro 9, by BD (2 mentions)
Facscan, by BD (2 mentions)
Hla dr pe, by Immunotools (2 mentions)
Human insulin, by Merck Group (2 mentions)
Cd86 fitc, by Immunotools (2 mentions)
Hla dr fitc, 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)
Cd90 fitc, by BioLegend (1 mentions)
Triton x 100, by Avantor (1 mentions)
Hla dr v500, by BD (1 mentions)
Anti human cd34 pe, by BD (1 mentions)
5 protocols using hla abc fitc
1
Characterization of Mesenchymal Stem Cells
2
Phenotyping of CD34+ HSCs Exposed to Glucocorticoids
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The phenotype of CD34+ HSC exposed to betamethasone or fluticasone after 20 h of culture was determined by flow cytometry. To that end, 20.000 CD34+ cells of each group (control, betamethasone, and fluticasone) were stained with anti-human CD34 PE (BD Biosciences), HLA-ABC FITC (Immunotools), CD45 PercP (BD Biosciences), CD40 APC (Immunotools), CD54 PE-Cy7, CXCR4 APC-Cy7 and HLA-DR V500 (BD Biosciences), and median fluorescence intensity values were obtained using FACS Canto II cytometer (BD Biosciences). Data were analyzed using FlowJo software (Tree Star Inc., Ashland, OR, USA).
3
Immunomodulatory Effects of PSAB-liposomes and Liraglutide on Dendritic Cells in Type 1 Diabetes
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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.
4
Characterization of hAF-MSC Surface Markers
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To characterize hAF-MSCs, the cell surface markers of cells cultured in basic media containing 10% FBS or 10% hPL were evaluated. The cells were trypsinized with 0.25% trypsin-EDTA at 37°C for 1 min and centrifuged at 2,035 × g for 6 min at room temperature to obtain the cell pellets. Then, non-specific binding was blocked using 10% human AB serum [serum from type AB donors; processed by our laboratory and inactivated at 53°C for 90 min as previously described (16 (link))] at 4°C for 30 min. Subsequently, they were incubated with the following monoclonal antibodies: Mouse anti-human CD34-FITC (cat. no. 343504; BioLegend, Inc.), CD44-FITC (cat. no. 21810443; ImmunoTools GmbH), CD45-phycoerythrin (PE; cat. no. 304008; BioLegend, Inc.), CD73-PE (cat. no. 21270734; ImmunoTools GmbH), HLA-ABC-FITC (cat. no. 21159033; ImmunoTools GmbH) and HLA-DR-PE (cat. no. 21388994; ImmunoTools GmbH). Cell surface marker expression was detected using a FACScan (BD Biosciences) and analyzed using CellQuest™ Pro 9.0 software (BD Biosciences).
5
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).
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