Peripheral blood mononuclear cell (PBMC) were labeled with carboxyfluorescein succinimidyl ester (CFSE, 200 nM; interchim, Montluçon, France) and seeded in 96-well round-bottom plates at 2.105/well. Cells were cultured in RPMI 1640 supplemented with 10% human AB serum (Biowest, Nuaillé, France) and anti-CD3 and anti-CD28 monoclonal antibodies (0.6 µg/mL, Sanquin, Amsterdam, The Netherlands). After 4 days of culture, cells were harvested and labeled with CD2 PC7, CD8 APC (Beckman Coulter, Miami, FL), CD4 BUV496, CD14 BV605 (Becton Dickinson, San Jose, CA, USA) and DAPI (Sigma-Aldrich, St Louis, MO, USA). CFSE dilution was assessed on DAPIneg viable T-cells by flow cytometry on Fortessa X-20 (Becton Dickinson) and the results were analyzed with ModFit LT software.
Cd14 bv605
Manufactured by BD
Sourced in United States
CD14-BV605 is a fluorochrome-conjugated antibody that binds to the CD14 cell surface antigen. CD14 is a marker for monocytes and macrophages. The BV605 fluorochrome is used for flow cytometry applications.
Automatically generated - may contain errors
Lab products found in correlation
Human ab serum, by Biowest (2 mentions)
Cd4 buv496, by BD (2 mentions)
Carboxyfluorescein succinimidyl ester (cfse), by BD (2 mentions)
Fortessa x20, by BD (2 mentions)
Compensation bead particles, by BD (2 mentions)
Cd4 buv737, by BD (2 mentions)
Lsr 2 flow cytometer, by BD (2 mentions)
Rpmi 1640, by Biowest (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Coptisine, by Merck Group (1 mentions)
D arginine, by Merck Group (1 mentions)
Fvs780, by BD (1 mentions)
L arginine, by Merck Group (1 mentions)
Horizon brilliant stain buffer, by BD (1 mentions)
Cd45 apc h7, by BD (1 mentions)
Cd4 bv510, by BD (1 mentions)
Falcon round bottom polystyrene tubes, by Corning (1 mentions)
Cd3 percp clone sk7, by BD (1 mentions)
Cd8 pe clone hit8a, by BD (1 mentions)
Cd25 pe cf594, by BD (1 mentions)
6 protocols using cd14 bv605
1
CFSE-based Proliferation Assay for T-cell Activation
2
T Cell Proliferation Assay with CFSE
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PBMC isolated after Ficoll density gradient were labeled with carboxyfluorescein succinimidyl ester (CFSE, 200 nM; interchim, Montluçon, France). The quantification of T cells was obtained by staining PBMC with an anti-CD3 APC antibody (Becton Dickinson). PBMC were seeded in 96-well round-bottom plates at a concentration of 1 × 105 T cells per well. Cells were cultured in RPMI 1640 supplemented with 10% human AB serum (Biowest, Nuaillé, France) and anti-CD3 and anti-CD28 monoclonal antibodies (0.6 μg/mL, Sanquin, Amsterdam, Netherlands). When indicated, cells were cultured in presence of the following chemical inhibitors or their controls: (i) L-arginine (1 mM, Sigma-Aldrich, St Louis, MO, USA) or with control D-arginine; (ii) coptisine (an IDO inhibitor) (50 nM, Sigma-Aldrich, St Louis, MO, USA) or vehicle; and (iii) PD-L1 blocking antibodies (10 μM, ebioscience, San Diego, CA, USA) or IgG. After 4 days of culture, cells were harvested and labeled with FVS 780 (Becton Dickinson) for cell viability, CD2 PC7 and CD8 APC (Beckman Coulter), CD4 BUV496 and CD14 BV605 (Becton Dickinson). CFSE dilution was assessed on FVSneg viable T cells by flow cytometry on Fortessa X20 (Becton Dickinson) and results were analyzed with ModFit LT software (Verity Software, Topsham, ME).
3
Comprehensive Immunophenotyping of Peripheral Blood
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PB samples were collected through venipuncture in 2 mL BD Vacutainer® spray-coated K2EDTA blood collection tubes (BD Biosciences, San Jose, CA, USA; #367841). One hundred μL of whole blood was transported into 5 mL Corning™ Falcon™ Round-Bottom Polystyrene Tubes (#352054) and stained with 12 monoclonal antibodies against the surface markers: CD45-APC-H7 (clone 2D1, #560178); CD3-PerCP (clone SK7, #340663); CD4-BV510 (clone SK3; #562970); CD8-PE (clone HIT8a, #555635); CD14-BV605 (clone M5E2, #564054); CD16-APC (clone B73.1, #561304); CD56-APC-R700 (clone NCAM16.2, #565139); CD25-PE-CF594 (clone M-A25, #562403); CD11b-BV786 (clone D12, #742642); CD183 (CXCR3)-BV421 (clone 1C6/CXCR3, #562558); CD194 (CCR4)-BV650 (clone 1G1, #744140); and CD196 (CCR6)-BB515 (clone 11A9, #564479) (all from BD Biosciences). The staining procedure was performed in BD Horizon™ Brilliant Stain Buffer (BD Biosciences, #563794) followed by red blood cells lysis with 1x BD FACS™ lysing solution (BD Biosciences, #349202) as suggested by the manufacturer for the Lyse-no-Wash protocol.
4
Phenotyping PBMCs Activated by LPS
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PBMCs (1 × 106/well) were stimulated overnight at 37°C with LPS 10μg/ml (E. coli serotype O55:B5; Alexis Biochemicals). After 1 h, 5μg/ml of brefeldin (Sigma-Aldrich) was added. After 12 hours of stimulation, cell surface staining was performed with CD14-BV605, HLA-DR BV650, CD16-APCH7, CD3-AF700 (BD Biosciences San Jose,CA). After washes in PBS, cells were fixed in Cytofix/Cytoperm buffer (BD Biosciences), for 30 min at 4°C, washed in PermWash buffer (BD Biosciences, and then stained for intra-cellular markers: IL8-BV510, TGFβ-PE-CF594, TNFα-APC (BD Biosciences San Jose, CA); IL10-AF488 (R&D systems); IL1β-PE (eBioscience, San Diego,CA); IL6-PECy7 (Biolegend, San Diego, CA) for 30 min at 4°C. After wash, PBMCs were resuspended in PBS, before their flow cytometry acquisition on LSR2 flow cytometer. Data were analyzed using FlowJo v9 (Tree Star, Inc.) and DIVA softwares (BD Biosciences).
5
Immunophenotyping of PBMC subsets following VitD3 treatment
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To identify specific immune cell subsets in PBMCs following VitD3 treatment, cells were stained with fluorescently-conjugated monoclonal antibodies; CD4-BUV737, CD45RO-APC, CD161-FITC, CD194-V450, CD196-PE, CD14-BV605, CD19-APC-H7, CD56-BV421, CD282-AF647 (TLR2), CD284-BV786 (TLR4; all from BD Bioscience; San Diego, CA, USA), and anti-TLR7-PE (Gibco Life Technologies, Carlsbad, USA), Zombie Aqua™ Fixable Viability Kit (BioLegend, San Diego, USA). Compensation bead particles were used to account for spectral overlap (BD Bioscience, San Diego, CA, USA) and analyzed using the BD LSRII flow cytometer. Unstained PBMCs and fluorescence minus one (FMO) were used as controls and a minimum of 100,000 events were analyzed per sample gated on live, single cell lymphocyte gate based on FSC and SSC, where the expression of the cell surface molecules was evaluated using FlowJo, LLC v10.4.2 software. Refer to Supplementary Figures 1–4 for gating strategies.
6
Identification of Immune Cell Subsets in PBMCs
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To identify specific immune cell subsets in PBMCs following 1,25(OH)2D3 treatment, the cells were stained with fluorescently-conjugated monoclonal antibodies CD4-BUV737, CD14-BV605, and CD3-BV510 (BD Bioscience; San Diego, CA, USA). Compensation bead particles were used to account for the spectral overlap (BD Bioscience, San Diego, CA, USA) above and analyzed using the BD LSRII flow cytometer. Unstained PBMCs were used as a control and a minimum of 20,000 events were analysed per sample gated on live, single cell lymphocyte gate based on FFS and SSC, where the expression of the cell surface molecules were evaluated using MFI values using BD FACSDiva 8.0.1 software (Becton, Dickinson and Company, Franklin Lakes, NJ, USA).
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