To study the surface cell markers on monocytes (CD14 +), PBMCs from the 10 patients used for single-cell analysis and 10 HDs were defrosted and washed once with PBS. After blocking for non-specific binding with Fc block (BD Pharmingen) for 5 min on ice, cells were incubated for 20 min on ice using staining buffer (PBS with 4% fetal bovine serum and 0.4% EDTA). Antibodies used included the following: CD14-FitC (Miltenyi Biotec), CD85-PEvio770 (Miltenyi Biotec), CD172a-APC (Miltenyi Biotec), CD97-PEvio770 (Miltenyi Biotec), CD31-PE (Miltenyi Biotec), CD366-PEvio615 (Miltenyi Biotec), CD62L-APC (Miltenyi Biotec), CD58-PE (Miltenyi Biotec), CD191-PEvio770 (Miltenyi Biotec), CD52-PEvio615 (Miltenyi Biotec), CD48-APC (Miltenyi Biotec). Cells were analyzed in a BD FACSCanto-II flow cytometer.
Cd14 fitc
Manufactured by Miltenyi Biotec
Sourced in United States, United Kingdom
CD14-FITC is a fluorochrome-conjugated antibody that binds to the CD14 antigen, which is expressed on the surface of monocytes and macrophages. It is used in flow cytometry applications for the identification and enumeration of these cell populations.
Automatically generated - may contain errors
Lab products found in correlation
Cd86 apc, by Miltenyi Biotec (3 mentions)
Facscanto 2 flow cytometer, by BD (2 mentions)
Fc block, by BD (2 mentions)
Facscalibur, by BD (2 mentions)
Cd3 percp, by Miltenyi Biotec (2 mentions)
Cd8 fitc, by Miltenyi Biotec (2 mentions)
Cd14 microbead, by Miltenyi Biotec (2 mentions)
Interleukin 4 (il 4), by Miltenyi Biotec (2 mentions)
Gm csf, by Miltenyi Biotec (2 mentions)
Cd31 pe, by Miltenyi Biotec (1 mentions)
Cd62l apc, by Miltenyi Biotec (1 mentions)
Cellquest software, by BD (1 mentions)
Ionomycin, by Merck Group (1 mentions)
Brefeldin a, by Merck Group (1 mentions)
Il 17 pe, by Miltenyi Biotec (1 mentions)
Cd4 pe, by Miltenyi Biotec (1 mentions)
Lsm710 confocal microscopy, by Zeiss (1 mentions)
Cd3 v500, by BD (1 mentions)
Cd19 pe cy7, by BD (1 mentions)
Zen imaging software, by Zeiss (1 mentions)
11 protocols using cd14 fitc
1
Profiling Monocyte Surface Markers
2
Intracellular Cytokine Production in CD4+ T Cells
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After separation with microbeads, CD4+ T cells were stained with mouse anti-human monoclonal antibodies: CD14-Fitc or CD8-Fitc, CD4-Pe, CD3-PercP and the isotype control (Miltenyi Biotec) and analyzed by flow cytometry to assess purity.
The analysis of intracellular cytokine production was done after 12 days of culture; T cells were stimulated for 6 hours with phorbol 12-myristate 13-acetate (PMA, 10 ng/mL), Ionomycin (500 ng/mL) and Brefeldin A (BFA 10 μg/mL) (Sigma Aldrich) and the intracellular staining was performed incubating T cells with mouse monoclonal antibody IL-17Pe (Miltenyi Biotec), T cells were acquired on a FACScalibur (BD Biosciences) and analyzed with Cell Quest software (BD Biosciences).
The analysis of intracellular cytokine production was done after 12 days of culture; T cells were stimulated for 6 hours with phorbol 12-myristate 13-acetate (PMA, 10 ng/mL), Ionomycin (500 ng/mL) and Brefeldin A (BFA 10 μg/mL) (Sigma Aldrich) and the intracellular staining was performed incubating T cells with mouse monoclonal antibody IL-17Pe (Miltenyi Biotec), T cells were acquired on a FACScalibur (BD Biosciences) and analyzed with Cell Quest software (BD Biosciences).
3
Internalization of MSC-Derived EVs by IECs
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To assess EV internalization by IECs, MSC membranes were stained with 2×10−6 M of PKH26 PKH26 Red Fluorescent dye (Sigma-Aldrich) according to manufacturer’s recommendations. Then, PKH26-labeled or -unlabeled MSCs were cultured in presence of IECs and EV uptake was assessed after 1, 2 or 4 days. At the end of co-culture, cells were detached by trypsin and stained with the following monoclonal antibodies: CD45-Vioblue (Miltenyi Biotec), CD3-V500 (BD Biosciences), CD4-APC-Vio770, CD8-FITC, CD14-FITC (Miltenyi Biotec), CD16-PercP-Cy5, CD19-PE-Cy7 (BD Biosciences) to identify the different IEC population, while TOPRO-3 was used to identify viable cells. The internalization of MSC-derived EVs by IECs was analyzed by FACS analysis.
To further confirm the transfer of EVs into IECs, cells were analyzed at the end of co-culture by confocal microscopy. Briefly, cells were detached by trypsin and stained with Viobright-FITC anti-human CD45 monoclonal antibody (Miltenyi Biotec). Then, cells were fixed using Cytofix/Cytoperm kit (BD Biosciences) and TOPRO-3 (Invitrogen Life Technologies) was used to reveal nuclei. Finally, cells were loaded into the CytoSpin centrifuge’s sample chamber and centrifuged 5 minutes at 400 rpm.
Images were obtained by LSM 710 confocal microscopy (Zeiss) at 63x magnification and elaborated with ZEN imaging software (Zeiss).
To further confirm the transfer of EVs into IECs, cells were analyzed at the end of co-culture by confocal microscopy. Briefly, cells were detached by trypsin and stained with Viobright-FITC anti-human CD45 monoclonal antibody (Miltenyi Biotec). Then, cells were fixed using Cytofix/Cytoperm kit (BD Biosciences) and TOPRO-3 (Invitrogen Life Technologies) was used to reveal nuclei. Finally, cells were loaded into the CytoSpin centrifuge’s sample chamber and centrifuged 5 minutes at 400 rpm.
Images were obtained by LSM 710 confocal microscopy (Zeiss) at 63x magnification and elaborated with ZEN imaging software (Zeiss).
4
Imaging of Apoptosis in THP-1 Cells
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THP-1 monocytic cells were stained with LysoTracker Red (Life Technologies), MitoTracker Green (Life Technologies), Hoechst 33342 (Life Technologies) and CD14-FITC (Miltenyi Biotec), according to manufacturer's instructions, before time-lapse imaging or induction of apoptosis. THP-1 cells were transfected with non-targeted GFP, Mito-DsRed (a kind gift from Dr Laura Osellame and Professor Mike Ryan), GFP-histone H2B and GFP-histone H4 (a kind gift from Dr Kylie Wagstaff and Professor David Jans) via Nucleofection 4D in accordance with the manufacturer's instructions. Confocal microscopy was performed at 37 °C in a humidified atmosphere with 5% CO2 using either the Zeiss Spinning Disk or the Zeiss LSM780 confocal microscope (Zeiss).
5
Evaluating Dendritic Cell Activation
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After five days in culture, surface staining was performed on monocyte-derived dendritic cells (MoDCs) for flow cytometry analysis with mouse anti-human HLA-DR FITC, CD83 PE, CD86 APC, CD54 PeVio770, CD14 FITC, CD3 PercP (all from Miltenyi Biotech) and analyzed by flow-cytometer LSRFortessa™ X-20 cell analyzer (BD Bioscience, Frankin Lake, NJ, USA) according to standard protocol. MoDC were then incubated with natural and synthetic compound in 12-well plates at concentration of 8 × 105 cell/mL. Stimulation with PAM2CSK4 1 µg/mL (Invivogen, San Diego, CA, USA) was used as positive control. Cells treated only with vehicle (DMSO) were used as the control. For the experiment with the anti TLR-4 neutralizing antibody, cells were incubated 30 min with only the antibody (1 µg/mL) at 37 °C before addition of PGDG. After 24 h, expression of all surface markers was estimated again by staining with fluorochrome-conjugated antibodies.
6
Multiparametric Flow Cytometry of ACE-2
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Cells (1.106 cells/well) were suspended in PBS containing 5% FBS and 2mM EDTA (Sigma-Aldrich). Suspended cells were incubated with viability dye (Live/Dead Near IR, Invitrogen), CD14-FITC, anti-ACE-2-PE or appropriate isotype control (Miltenyi) for 30 minutes at 4°C. Labelled cells were then permeabilized using BD Cytofix/Cytoperm kit and stained with CD68-PE-Cy7 (Miltenyi). Data were collected on a Navios instrument (Beckman Coulter) and analyzed with FlowJo software (FlowJo v10.6.2, Ashland, OR).
7
Generating Mature Dendritic Cells
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Peripheral blood mononuclear cells (PBMCs) from human healthy donors were thawed from ImmunXperts SA (Belgium) biobank. Monocytes were isolated from PBMCs using a MACS magnetic separation column CD14 MicroBeads (Miltenyi) and purity was evaluated by CD14 FACS staining (Fortessa). Cells were then resuspended at a cellular density of 106 cells/mL and plated into a 24-well tissue culture microplate (1mL per well) in CellGenix DC medium (CellGenix, Cat.N° 20801-0500) added with Gentamycin, IL-4 (Miltenyi, 130-093-866) and GM-CSF (Miltenyi, 130-093-922) for 5 days. At day 5, cells were stained for FACS analysis with several DC activation markers to assess their immature dendritic cell (iDC) state: CD14-FITC (Miltenyi, 130-110-518), CD40-BV510 (BD Biosciences, 563456), CD80-BV421 (BD Biosciences, 564160), CD83-PE-Vio 770 (Miltenyi, 130-110-505), CD86-APC (Miltenyi, 130-116-161), CD209-PE (Miltenyi, 130-117-706), and HLA-DR-BUV395 (BD Biosciences, 564040). On the same day, respective antigens (10µg/mL) were added to the cell culture for 48h. At day 7, cells were stained for FACS analysis with same markers to assess their mature state. LPS (Sigma, L4391-1MG) (1µg/mL), and TNF (Miltenyi, 130-094-024) (800U/mL), with IL-1b (Miltenyi, 130-093-898) (150U/mL), were used as positive control.
8
Quantification of Granulocytic Myeloid-Derived Suppressor Cells
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Peripheral blood was collected using EDTA and Heparin-coated tubes and samples were shipped to the Universitätsklinik für Kinder-und Jugendmedizin, Tübingen (Germany) at room temperature and analyzed within 24 h. MDSCs were characterized as previously described (21 (link), 22 (link)). In brief, peripheral blood mononuclear cells (PBMCs) were isolated from whole blood by Ficoll density gradient centrifugation (Lymphocyte Separation Medium; Biochrom), washed with RPMI-1640 and cell viability was confirmed by trypan blue staining. The isolated PBMC, containing only low density granulocytes, were stained with specific antibodies for G-MDSC (CD66b-FITC, CD33-PE) and M-MDSC (CD14-FITC and HLADR-PerCP) (Miltenyi Biotec) and quantified by flow cytometry using a FACSCalibur (BD). G-MDSCs were phenotypically characterized as low-density fraction granulocytes CD33+CD66b+ cells (Figure 1 ). The percentage of G-MDSC was determined as ratio of CD33+CD66b+ cells (P2 in Figure 1 ) over total PBMCs containing the low density granulocyte fraction (P1 in Figure 1 ). Calculations were performed with BD CellQuest Pro analysis software and FlowJo V7.
9
Differentiation and Activation of Human moDCs
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Peripheral blood
mononuclear cells were isolated
from buffy coats obtained from HLA-A2.1+ healthy volunteers
after written informed consent and in agreement with institutional
guidelines using Ficoll density gradient centrifugation (Lymphoprep,
ELITechGroup). CD14+ monocytes and naïve CD8+ T cells were isolated using CD14 microbeads and the human
Naive CD8+ T cell isolation kit (both Miltenyi Biotec),
respectively. Monocytes were differentiated into immature monocyte-derived
DCs (moDCs) in 6 days using RPMI 1640 containing 10% FCS, 2 mMl -glutamine, 0.5% antibiotic–antimycotic enriched with
interleukin-4 (IL-4, 300 U/mL), and GM-CSF (450 U/mL) (both Miltenyi
Biotec). Isolated cells were stored in liquid nitrogen in 10% DMSO
(CryoSure) and 90% FBS.
Human moDCs were thawed from liquid
nitrogen. In vitro DC activation (24 and 48 h) in
alginate cryogels containing NY-ESO-1 PLGA NPs was studied, as described
for BMDCs. Soluble NY-ESO-1 peptide and TLR ligands were added to
the controls in the same amounts as is present in NPs. Flow cytometric
staining was performed using Zombie Violet Fixable viability dye for
30 min at 4 °C, followed by cell-surface staining with the following
antibodies for 30 min at 4 °C: CD14-FITC (Miltenyi Biotec), CD80-PerCP-eFluor710
(PharMingen), CD86-PeCy7 (PharMingen), and CD40-PE (Beckman).
mononuclear cells were isolated
from buffy coats obtained from HLA-A2.1+ healthy volunteers
after written informed consent and in agreement with institutional
guidelines using Ficoll density gradient centrifugation (Lymphoprep,
ELITechGroup). CD14+ monocytes and naïve CD8+ T cells were isolated using CD14 microbeads and the human
Naive CD8+ T cell isolation kit (both Miltenyi Biotec),
respectively. Monocytes were differentiated into immature monocyte-derived
DCs (moDCs) in 6 days using RPMI 1640 containing 10% FCS, 2 mM
interleukin-4 (IL-4, 300 U/mL), and GM-CSF (450 U/mL) (both Miltenyi
Biotec). Isolated cells were stored in liquid nitrogen in 10% DMSO
(CryoSure) and 90% FBS.
Human moDCs were thawed from liquid
nitrogen. In vitro DC activation (24 and 48 h) in
alginate cryogels containing NY-ESO-1 PLGA NPs was studied, as described
for BMDCs. Soluble NY-ESO-1 peptide and TLR ligands were added to
the controls in the same amounts as is present in NPs. Flow cytometric
staining was performed using Zombie Violet Fixable viability dye for
30 min at 4 °C, followed by cell-surface staining with the following
antibodies for 30 min at 4 °C: CD14-FITC (Miltenyi Biotec), CD80-PerCP-eFluor710
(PharMingen), CD86-PeCy7 (PharMingen), and CD40-PE (Beckman).
10
Immunophenotyping of Differentiated Cells
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For the study of surface cell markers, cells were harvested after differentiation culture and washed once with PBS. Cell staining was performed in a staining buffer (PBS with 4% fetal bovine serum and 0.4% EDTA) after blocking for non-specific binding with Fc block (BD Pharmingen) for 5 minutes on ice. Cells were stained for 20 minutes on ice. Antibodies used included: CD14-FITC, CD80-PE, CD86-APC (Miltenyi biotec), CD11b-APC, CD1a-PE (Biolegend), HLA-DR-PeCy7 (eBioscience). Cells were also stained with the viability dye LIVE/DEAD TM Fixable Violet (Invitrogen) according to manuacturer's conditions. After staining, cells were fixed with PBS + 4% paraformaldehyde and analyzed in a BD FACSCanto-II flow cytometer in the following 48 h.
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