The study was approved by the Institutional Review Board of the NIH and conformed to the tenets of the Declaration of Helsinki. Human blood samples from four healthy individuals were obtained from the NIH blood bank. Human PBMCs were isolated from the blood of healthy donors using a Ficoll gradient centrifugation protocol. PBMCs were then treated with buffer vehicle, LPS (5 μg/ml) or GroEL (5 μg/ml) for 24 h, followed by anti-CD14-APC, anti-CD16-FITC and anti-CD163-PE staining (BD Biosciences, San Jose, CA, USA). Fluorescent cells were acquired on a flow cytometer (BD FACSCalibur, San Jose, CA, USA) and analysed using the FlowJo software (V10, Tree Star, Ashland, OR, USA).
Anti cd14 apc
Manufactured by BD
Sourced in United States
Anti-CD14-APC is a fluorescently labeled antibody that binds to the CD14 receptor expressed on the surface of monocytes and macrophages. It is a useful tool for the identification and analysis of these cell types in flow cytometry applications.
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Lab products found in correlation
Facscalibur, by BD (4 mentions)
Anti cd163 pe, by BD (3 mentions)
Anti cd16 pe, by BD (3 mentions)
Anti cd3 pe cy7, by BD (3 mentions)
Anti cd11c pe, by BD (3 mentions)
Anti cd19 pe, by BD (3 mentions)
Anti cd16 fitc, by BD (2 mentions)
Anti cd80 pe, by BD (2 mentions)
Anti cd45 fitc, by BD (2 mentions)
Anti cd3 fitc, by BD (2 mentions)
Pe cf594 anti cd163, by BD (2 mentions)
Anti cd80 bb515, by BD (2 mentions)
Anti hla dr percp cy5, by BD (2 mentions)
Facsdiva software, by BD (2 mentions)
Facscalibur cytometer, by BD (2 mentions)
Anti cd11b apc, by BD (1 mentions)
Attune next, by Thermo Fisher Scientific (1 mentions)
Anti cd45 percp cy5, by BD (1 mentions)
Anti cd15 apc, by BD (1 mentions)
Anti cd3 bv421, by BD (1 mentions)
28 protocols using anti cd14 apc
1
Characterization of Human Immune Cells
2
Flow Cytometric Immune Cell Profiling
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Cells were washed and stained with Fixable Viability Stain eFluor 780 (Thermo Fisher Scientific), anti-CD45 PerCPCY5.5, anti-GPR56 PE, anti-CD11b APC, anti-CD14 APC, anti-CD15 APC, anti-CD3 BV421 and anti-CD19 BV421 (all BD Biosciences, Le Pont de Claix) and processed on an Attune Next (Thermo Fischer Scientific) flow cytometer. Cell counts were obtained after manual gating on FlowJo V10.6.2 (Beckton Dickinson, Le Pont de Claix). Details are provided in the Supplementary Appendix.
3
Leukocyte Surface Marker Analysis
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The authors collected blood samples from the antecubital fossa vein. For the surface staining of leukocytes, 50 µL of anticoagulated blood was added to tubes containing 5 µL of fluorochrome-labeled monoclonal antibodies, including anti-CD11b allophycocyanin (APC), clone Bear1; anti-CD14-APC, clone M5E2; anti-CD15 fluorescein isothiocyanate (FITC), clone 80H5; anti-CD16 R-phycoerythrin (RPE), clone 3G8; anti-CD62L-RPE, clone DREG56; anti-CD64-RPE, clone 22; anti-CD163-FITC, clone GHI/61; and anti-CD274-APC, clone PD-L1. The anti-CD163-FITC and anti-CD14-APC were manufactured by BD Biosciences (Franklin Lakes, NJ, USA), and all the other antibodies were manufactured by Beckman Coulter (Miami, FL, USA). The blood samples were incubated with the antibodies for 15 min at room temperature in the dark. Then, a lysing solution (OptiLyse C, Beckman Coulter, Miami, FL, USA) was added, and the mixture was incubated for another 10 min. The flow cytometric evaluation was conducted with a Navios 10 flow cytometer (Beckman Coulter, Miami, FL, USA). The gating strategy for the granulocytes and monocytes is presented in Figure 1 .
4
Immunophenotyping of Immune Cells
5
Mesenchymal Stem Cell Immunophenotyping
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MSCs were trypsinized for identification of several surface markers using human anti-CD29-PE, anti-CD44-V450A, anti-CD105-FITC, anti-CD45-FITC, anti-CD14-APC and anti-HLA-DR-PE antibodies (all purchased from BD Pharmingen, Franklin Lakes, NJ, USA). Flow cytometry was performed using a BD Influx cell sorter (BD Biosciences, Franklin Lakes, NJ, USA).
6
Multiparametric flow cytometry analysis
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Cell surface molecules were analysed by a FACSCalibur® flow cytometer (Becton Dickinson). Cells were incubated for 10 min at 4°C with relevant or control antibodies in 100 µl total staining volume in PBS-BSA. After staining, the cells were washed and resuspended in 300 µl of PBS-BSA. The following antibodies were used for the staining of human monocytes, T cells and dendritic cells:, anti-CD1c-FITC [anti-BDCA-1] (Miltenyi), anti-CD11b-PE [Mac-1] (BD Pharmingen), anti-CD14-APC (BD Pharmingen), anti-CD40-FITC (BD Pharmingen), anti-CD45RA-APC (BD Pharmingen), anti-CD86-PE (eBioscience), anti-HLA-DR-CyChrome (BD Pharmingen). The following antibodies were used for the staining of murine dendritic cells and T cells: CD4-AF647, vα2-Bio, SA-PacificBlue, CD11c-FITC, CD86-PE, MHC II was measured by a combination of biotin-labeled I-Ab/SA-APC (all BD Pharmingen). To measure proliferation, T cells were labeled with the fluorescent dye CFSE (Molecular Probes).
7
Isolation of Monocyte Subsets by FACS
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The CD14 + + CD16-, CD14 + + CD16 + , and CD14 + CD16 + + monocyte subsets were isolated from whole population of monocytes by cell sorting (FACS Aria cell sorter, BD Biosciences Immunocytometry Systems, San Jose CA) after labeling with anti- CD14 APC and anti-CD16 PE (BD Pharmingen, San Diego CA).
8
Chemotaxis Assay for Immune Cells
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PQ-primed A549 cell supernatants enriched for mtDNA (approximately 104 copies/μL) were treated with or without DNaseI for the chemotaxis assay. Whole blood samples were obtained from healthy volunteers. Cells were isolated immediately by a one-step gradient centrifugation method using Polymorphprep reagent according to the described protocol [19 (link)]. The chemotactic responses of PBMCs and PMNs were assessed by transwell cell culture chambers with polycarbonate filters with 5 μm pores. The fold chemotaxis index was calculated by dividing the number of cells migrating in the presence of supernatants by those migrating toward medium alone. Anti-CD14-APC, anti-CD3-FITC, and anti-CD19-PE (BD Biosciences, San Jose, CA) were used to distinguish the subtypes of the migrated cells by flow cytometry.
9
Immune Cell Analysis of COVID-19 Vaccinated Donors
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Human PBMCs collected from likely COVID-19-vaccinated donors between February 2022 and February 2023 were isolated from buffy coats by means of Ficoll density gradient centrifugation and infected for 24 h with 5 MOI of Prime-2-CoV_Beta (SPEk103) or left untreated. Cells were collected by scraping, washed in PBS and incubated for 30 min at 4 °C with the following antibody cocktail in PBS: anti-CD14 APC (BD Biosciences, San Jose, CA, USA, 561383), anti-CD4 BV605 (BD Biosciences, 562843), anti-CD8 APC-H7 (BD Biosciences, 561423), anti-CD19 BV786 (BD Biosciences, 563325), anti-CD3 PE-Cy7 (BD Biosciences, 557749), anti-CD56 PE (BD Biosciences, 556647), anti-CD25 BV421 (BD Biosciences, 567485), LIVE/DEAD™ Fixable Aqua stain (Invitrogen, Waltham, MA, USA, L34957). After three washes in PBS, cells were fixed in 4% formaldehyde for 20 min, washed and finally resuspended in FACS buffer. Data were acquired with an Attune NxT cytometer (Thermo Fisher Scientific). Data analysis was performed using Kaluza software (version 2.1, Beckman Coulter, Brea, CA, USA).
10
Phenotypic Analysis of Polarized HMDMs
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HMDMs were isolated, differentiated, and polarized as described above for 24 h. Polarized cells were treated with 50 nM thioA for 7 h. Cells were detached using accutase solution (Sigma-Aldrich #A6964), washed, and resuspended in FACS wash (PBS, 2.5% FCS, 0.1% sodium azide). Cells were blocked in human Fc Block (BD, #564220) for 15 min, and stained for 30 min on ice with anti-CD14-APC (BD #555399), anti-CD163-PE-CF594 (BD #562670), anti-CD80-BB515 (BD #565008), and anti-HLA-DR-PerCP-CY5.5 (BD #560652) antibodies. After washing, stained cells were resuspended in 1% paraformaldehyde in PBS prior to flow cytometric analysis on a BD LSRFortessa. Data were analyzed using BD FACSDiva software (BD Biosciences, San Jose, CA, USA). Median fluorescence intensity of singlet cells was used to quantify surface marker expression.
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