1,25D3, 1,25D2 and analogues were manufactured at the Pharmaceutical Research Institute (Warsaw, Poland). The compounds were placed in glass ampoules at and kept −20 °C. Analogues were dissolved in absolute ethanol at 100 μM and further diluted in culture medium for each required experimental concentration. Antibodies for flow cytometry CD14-PE and CD11b-FITC were from ImmunoTools (Friesoythe, Germany). Antibodies for Western blotting and chemiluminescence blotting substrate were from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA).
Cd14 pe
Manufactured by Immunotools
Sourced in United States, Germany
CD14-PE is a flow cytometry reagent that binds to the CD14 surface antigen, which is expressed on monocytes and macrophages. It is conjugated to the fluorescent dye phycoerythrin (PE) to enable detection and quantification of CD14-positive cells using flow cytometry.
Automatically generated - may contain errors
Lab products found in correlation
Cd11b fitc, by Immunotools (2 mentions)
Human insulin, by Merck Group (2 mentions)
Cd86 fitc, by Immunotools (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)
Il 1β, by Immunotools (2 mentions)
Hla abc fitc, by Immunotools (2 mentions)
Fcs express, by De Novo Software (1 mentions)
Accuri c6, by BD (1 mentions)
Fetal bovine serum (fbs), by Merck Group (1 mentions)
Penicillin, by Merck Group (1 mentions)
Streptomycin, by Merck Group (1 mentions)
L glutamine, by Merck Group (1 mentions)
7 protocols using cd14 pe
1
Synthesis and Characterization of Vitamin D Analogues
2
Hypomethylating Agents Induce Differentiation of Leukemic Cells
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HL60 and Jurkat cells were acquired from the local cell bank at the Institute of Immunology and Experimental Therapy in Wrocław (Poland), while KG1 cells were purchased from the German Resource Center for Biological Material (DSMZ GmbH, Braunschweig, Germany). The cells were cultured in RPMI-1640 medium (Biowest, Nuaillé, France) with 10% FBS, 2 mM L-glutamine, 100 units/mL penicillin, and 100 μg/mL streptomycin (all from Sigma-Aldrich) and maintained at standard cell culture conditions. DNA from AML patients’ blasts were kept frozen at −80 °C from the study published before [18 (link)].
In order to study the influence of hypomethylating agents, HL60 cells were exposed to 10 nM 1,25D, 1 μM ATRA, 50 or 100 μM zebularine (ZEB), and/or 5 or 10 μM 5-azacytidine (5AZA) for 96 h. The expression of cell surface markers of differentiation was determined by flow cytometry. Cells were washed and stained with antibodies: CD11b-FITC and CD14-PE (ImmunoTools) for 1 h on ice. Next, they were washed with ice-cold PBS and suspended in 0.5 mL of PBS supplemented with 0.1% bovine serum albumin (BSA) prior to analysis on Accuri C6 (Becton–Dickinson). Data analysis was performed using FCS Express (De Novo™ Software, Pasadena, CA, USA).
In order to study the influence of hypomethylating agents, HL60 cells were exposed to 10 nM 1,25D, 1 μM ATRA, 50 or 100 μM zebularine (ZEB), and/or 5 or 10 μM 5-azacytidine (5AZA) for 96 h. The expression of cell surface markers of differentiation was determined by flow cytometry. Cells were washed and stained with antibodies: CD11b-FITC and CD14-PE (ImmunoTools) for 1 h on ice. Next, they were washed with ice-cold PBS and suspended in 0.5 mL of PBS supplemented with 0.1% bovine serum albumin (BSA) prior to analysis on Accuri C6 (Becton–Dickinson). Data analysis was performed using FCS Express (De Novo™ Software, Pasadena, CA, USA).
3
Immune Cell Phenotyping by Flow Cytometry
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Cells from human blood were labelled at day 0 after Ficoll-Hypaque density gradient centrifugation and at day 2, after stimulation with loaded MACSiBead, with the following antibodies: CD3-APC, CD4-PE (both BD Biosciences, CA, USA), CD69-FITC (Biolegend, CA, USA) and CD14-PE (ImmunoTools, Berlin, Germany). Labeled cells were sorted on a BD FACSMelody cell sorter. Cells were first gated for viability using the 7-AAD stain.
4
Synthesis and Preparation of 1,25D3 Analogs
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1,25D3 and all analogs were synthesized in the pharmaceutical research institute (Warsaw, Poland). The compounds were aliquoted and stored in glass ampoules under argon at −20°C. Compounds were dissolved in an absolute ethanol to 100 µM, and subsequently diluted in the culture medium to the required concentration. Antibodies CD14-PE and isotype control-PE were from ImmunoTools (Friesoythe, Germany).
5
Phenotypic Characterization of Macrophages
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After stimulation, MDMs were harvested using Trypsin-EDTA for 15 min followed by a cell scraper. Cells were washed once with ice-cold PBS and resuspended in ice-cold FACS buffer (0.5% BSA and 0.05% sodium azide in PBS). Cells (2 x 105 cells) were stained with the fluorochrome-conjugated anti-human antibodies, including CD80-FITC, CD163-PE, and CD206-FITC (BioLegend, San Diego, CA, USA), CD14-PE, CD16-FITC, and isotype control (ImmunoTools GmbH, Germany) for 30 min at 4°C in FACS buffer. The cells were then washed three times in ice-cold PBS and fixed with 2% formaldehyde (Sigma Aldrich, MO, USA) at 4°C for 10 min. After fixation, cells were washed once in PBS, resuspended in FACS buffer, and stored at 4°C in the dark until used. Cell surface expressions were detected using CytoFlex S (Beckman Coulter Life Sciences, Indiana, USA) and analyzed with CytoExpert software.
6
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.
7
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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