Five milliliters of CSF were obtained by lumbar spinal tap and centrifuged at 550g at 4°C for 5 minutes. The supernatant was removed, and the pellet was resuspended and incubated at 4°C for 20 minutes with fluorochrome-labeled antibodies against CD4, CD8, CD20, CD25, C-C chemokine receptor type 6 (CCR6), HLA-DR, CD14, and CD45 (BD Pharmingen Mouse Anti-Human Antibodies: CD4 PerCP, CD8 FITC, CD 20 Alexa Fluor 700, CD25 PE-Cy7, CD196 (CCR6) PE, Anti-HLA-DR V450, and CD14 APC; eBioscience mouse Anti-Human Antibody: CD45 EF 605). Cells were washed and resuspended in 200 μL of fluorescence-activated cell sorting (FACS) buffer before FACS analysis on a FACSCanto-II. Analysis of cell subset counts was performed with FlowJo. All CSF and serum tests were performed with researchers blinded to the clinical diagnosis and MRI results. CSF samples were measured by flow cytometry in a time frame of 90 minutes after lumbar spinal tab. CSF supernatants were stored in polypropylene tubes at −80°C until batched analysis of NfL and CHI3L1 concentrations.
Cd14 apc
Manufactured by Thermo Fisher Scientific
Sourced in United States
CD14-APC is a fluorescently labeled antibody that binds to the CD14 cell surface receptor. CD14 is a marker for monocytes and macrophages. The APC (Allophycocyanin) fluorophore allows for detection and analysis of CD14-positive cells using flow cytometry or other fluorescence-based techniques.
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Lab products found in correlation
Cd3 apc, by Thermo Fisher Scientific (4 mentions)
Cd8 fitc, by Thermo Fisher Scientific (2 mentions)
Igm pe cy5, by BD (2 mentions)
Cd16 apc, by Thermo Fisher Scientific (2 mentions)
Cd19 apc cy7, by BD (2 mentions)
Cd38 violet 421, by BD (2 mentions)
Cd27 pe, by BD (2 mentions)
Ficoll paque plus, by GE Healthcare (2 mentions)
Accuri c6, by BD (2 mentions)
Cd25 pe cy7, by Thermo Fisher Scientific (1 mentions)
Mouse anti human antibodies, by BD (1 mentions)
Cd4 percp, by Thermo Fisher Scientific (1 mentions)
Lsrii instrument, by BD (1 mentions)
Cd36 pe, by BioLegend (1 mentions)
Cd86 fitc, by BioLegend (1 mentions)
Cd38 pe, by BioLegend (1 mentions)
Ly6c fitc, by BioLegend (1 mentions)
F4 80 apc, by BioLegend (1 mentions)
Tlr4 apc, by BioLegend (1 mentions)
Ifn γ fitc, by BD (1 mentions)
18 protocols using cd14 apc
1
CSF Immune Cell Profiling by Flow Cytometry
2
Multimer-based peanut allergy analysis
3
Flow Cytometric Characterization of Cell Surface Markers
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For the detection of cell surface molecules, cells were incubated with the following antibodies: CD14 APC (eBioscience, Frankfurt, Germany), CD36 PE, CD38 PE, CD86 FITC, F4/80 APC, Ly6C FITC (all from BioLegend, Fell, Germany), or the appropriate isotype controls. Flow cytometric analysis was performed using FACSCalibur and analyzed using FlowJo software (Tree Star, Ashland, OR, USA).
4
Multiparameter Immune Cell Profiling
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CD3-percp-cy5.5 (Catalogue #45-0036-42), CD8-APC (Catalogue #17-0086-42), CD8-FITC (Catalogue #11-0086-42), CD14-APC (Catalogue #17-0149-42), CD56-FITC (Catalogue #4278380), CD16-PerCP-eFluor™710 (Catalogue #46-0168-42), CD11b-PerCP-eFluor™710 (Catalogue #46-0110-80), IFN-γ-PE (Catalogue #12-7319-42), TLR2-FITC (Catalogue #11-9922-41) and Mouse IgG1-PE (Catalogue #12-4714-81) were all bought from eBioscience. HLA-DR-PE (Catalogue #555812), IL-10-APC (Catalogue #554707), CXCR3-APC (Catalogue #550967) and IFN-γ-FITC (Catalogue #561053), Rat IgG2a-APC (Catalogue #554690) and mouse IgG-APC (Catalogue #5065947) were purchased from BD Biosciences. CD16-FITC (Catalogue #302006), HLA-DR-APC (Catalogue #307609), CD3-FITC (Catalogue #317306) and TLR4-APC (Catalogue #312815) were purchased from Biolegend. EP2-PE (Catalogue #10477) and Rabbit IgG-PE (Catalogue D5-1610) were purchased from Cayman Chemical.
5
Immunophenotyping of Mesenchymal Cells
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BM-MSC, fibroblasts or myofibroblasts were harvested using acutase, and incubated on ice for 1 h with directly conjugated antibodies including; SSEA-4-FITC (R&D Systems), CD44-PE (BD Bioscience), CD90-PE, CD105-APC, CD14-APC, CD45-PECy5 (eBioscience, Hatfield, UK), CD73-PE (BD Biosciences, Oxford, UK). Indirect staining was performed for GD2 (BD Biosciences), with a secondary goat anti-mouse-Alexa488 conjugated antibody at 1:200 for 40 min (Life Technologies). Cells were analysed using a FACScanto cytometer (Beckton Dickinson, Oxford, UK), and data analysed using FACS Diva (v6.2).
6
Pioglitazone Modulates Myelin Phagocytosis in Monocytes
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Monocytes were incubated with 1 μmol/L pioglitazone or dimethyl sulfoxide/phosphate buffered saline (DMSO/PBS) for 1 h at 37°C. Cells were stained with CD14‐APC (eBioscience, San Diego, CA, 17‐0149, 1:500) for 10 min at 37°C. Cells were resuspended in warm X‐vivo (Lonza). 10 μg/mL pHrodo‐labeled myelin was added to phagocytosing groups for 20 min at 37°C. Cold FACS buffer was then added and cells were analyzed immediately on a BD‐LSR II flow cytometer using BD‐FACSDiva 6.1 software (BD, Franklin Lakes, NJ). Gating for myelin was based on non‐phagocytosing controls and expression of surface markers was based on comparison of mean fluorescence intensity (MFI). Phagocytosis index = (FITC+ fluorescence in treated groups)/(FITC+ fluorescence in nonphagocytosing controls).
7
Multicolor Flow Cytometry Sorting of PBMCs
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PBMCs were stained with CD3-APC, CD19-APC, CD56-PE, and CD14-APC antibodies (eBioscience, State of California, USA) at 4°C for 30 minutes [28 (link)]. Isotype controls with irrelevant specificities were included as negative controls. Human TruStain FcX™ (Fc Receptor Blocking Solution, Biolegend, San Diego, California, USA) was used to block Fc-receptors. Cells were washed three times with sterilized phosphate-buffered saline (PBS). CD3+ T cells, CD19+ B cells, CD3−CD56+ NK cells, and CD14+ monocytes were gated and isolated with a MoFlo XDP high-speed flow cytometry sorter (Beckman, Brea, California, USA). The purified cells were collected for CNV analysis of the OR gene.
8
Pluripotency Analysis of Stem Cells
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Pluripotency analysis was performed with the BD Stemflow™ Human and Mouse Pluripotent Stem Cell Analysis Kit (BD Biosciences Oxford, UK). Specific surface markers were assessed with fluorochrome-labelled specific antibodies after blocking Fc non-specific interactions by incubation with an anti-CD16/32 antibody for 10–15 minutes. Parameters for analysis were set with unlabelled cells and gating with isotype controls for the appropriate fluorochromes. Compensation for spectral overlap was done when required using BD™ CompBead Plus positive and negative beads. Analysis was run on a LSFORTESSA BD cytometer using Flowjo (7.6.5). Antibodies: CD71-FITC; CD117-APC; CD41-FITC; CD33-PE (BD pharmingen); CD61-PE (Caltag); CD45-Pacblue (biolegend); CD34-FITC (Cambridge Bioscience); CD14-APC (ebioscience)
9
Isolation and Identification of Ara h 2-Specific B Cells
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PBMCs were isolated by density-gradient centrifugation (Ficoll-Paque Plus, GE Healthcare) and cryopreserved in FBS with 10% DMSO. After the study outcomes were assessed, PBMCs (10 × 106 to 25 × 106 cells per sample) were thawed and washed with PBS. Ara h 2–specific B cells were selected by flow cytometry using a fluorescent natural Ara h 2–Alexa Fluor 488 multimer, as previously described (7 (link)), using CD3-APC (eBioscience, clone OKT3), CD14-APC (eBioscience, clone 61D3), CD16-APC (eBioscience, clone CB16), CD19-APC-Cy7 (BD Biosciences, clone SJ25C1), CD27-PE (BD Pharmingen, clone M-T271), CD38–Violet 421 (BD Biosciences, clone HIT2), and IgM-PE-Cy5 (BD Pharmingen, clone G20-127). Single Ara h 2–specific B cells, identified as Ara h 2+CD3–CD14–CD16–CD19+CD27+ B cells, were index sorted into individual wells in a 96-well plate by FACS (Aria II or Fortessa, BD Biosciences) and stored at –80°C for single-cell B cell receptor (BCR) amplification. Data were analyzed using FlowJo software, version 8.8.7 (Tree Star).
10
Immunophenotyping of MM6 Monocytes
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About 1 × 105 MM6 cells were incubated for 30 min at room temperature with antibodies for CD11b (CD11b-PECy7, eBioscience), CD14 (CD14-APC, eBioscience), CD15 (CD15-eFluor 450, eBioscience), CD33 (CD33-PE P67.6, eBioscience), FVD eFluor® (eBioscience) or with the proper control IgGs. The optimal concentration for each antibody was adjusted according to manufacturer’s instructions. To block unspecific binding of antibodies, human Fc Block (Miltenyi) was added to the cell suspension. After washing with PBS, FACS analysis was performed using FACS Canto II (Becton Dickinson).
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