PBMCs were obtained by Ficoll–Hypaque density gradient centrifugation. If indicated, CD8+ or CD4+ T cells were further purified from isolated PBMCs by negative selection with magnetic beads using a CD8+ or CD4+ T cell Enrichment Kit (Cell purity >95%, Stem Cell Technologies, Vancouver, Canada). The following antibodies were used for immunostaining to isolate cell subtypes: FITC-CD3, APC-cy7-CD8, APC-CD4, PE-cy7-CD14 and 7-AAD (Biolegend, San Diego, CA, USA). CD4+ T cells (CD3+CD4+), CD8+ T cells (CD3+CD8+), and monocytes (CD3−CD14+) were selected from 7-AAD-negative live PBMCs using a FACSAria™ flow cytometer (BD Biosciences, Franklin Lake, NJ, USA).
Cd14 pe cy7
Manufactured by BioLegend
Sourced in United States
CD14 PE-Cy7 is a fluorochrome-conjugated monoclonal antibody that binds to the CD14 cell surface antigen. CD14 is a glycosylphosphatidylinositol (GPI)-anchored protein expressed primarily on the surface of monocytes and macrophages, and functions as a pattern recognition receptor that plays a role in the innate immune response.
Automatically generated - may contain errors
Lab products found in correlation
Cd3 pe cy7, by BioLegend (3 mentions)
Cd45 fitc, by BD (3 mentions)
Cd4 pe, by BioLegend (3 mentions)
Cd40 apc, by BioLegend (3 mentions)
Cd11a pe, by BioLegend (3 mentions)
7 aad, by BioLegend (2 mentions)
Cd3 fitc, by BioLegend (2 mentions)
Cd4 apc, by BioLegend (2 mentions)
Cd56 pe cy7, by BioLegend (2 mentions)
Cd107a pe, by BD (2 mentions)
Facscanto 2, by BD (2 mentions)
Cd3 apc cy7, by BD (2 mentions)
Cd56 apc, by BD (2 mentions)
Hla dr apc cy7, by BioLegend (2 mentions)
Cd14 fitc, by BioLegend (2 mentions)
Cd127 percp cy5, by BioLegend (2 mentions)
Cd45 apc cy7, by BioLegend (2 mentions)
Cd3 percp, by BioLegend (2 mentions)
Cd25 apc, by BioLegend (2 mentions)
Cd86 apc cy7, by BioLegend (2 mentions)
25 protocols using cd14 pe cy7
1
Isolation and Purification of PBMC Subsets
2
Evaluating macrophage M2 polarization
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To evaluate macrophage differentiation status, primary decidual macrophages and THP1 cells were stained with APC-conjugated anti-human CD163 antibody and PE-conjugated anti-human CD206 antibody for anti-inflammatory M2 bias. Macrophage purity was validated with PE-Cy7-CD14 (all from BioLegend, Beijing, China).
3
Multiparametric Immune Profiling of PBMCs
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PBMCs were thawed and washed twice with PBS. Then, the cells were stained with the following anti-human antibodies: PE-Cy7-CD3, PE/Dazzle594-CD4, APC-Cy7-CD8a, PE-Cy7-CD14, PerCP-Cy5.5-CD16, BV421-CD39, PE-CD73 (BioLegend), anti-nitrotyrosine (anti-NT) rabbit (Cat. #BS-8551R, Bioss Thermo Fisher) and Zombie Aqua (BioLegend). Nitric oxide production was evaluated using the molecular probe DAF-FM DA (10 μM, Cat. #D23844 Invitrogen). The oxidized product was measured at excitation/emission wavelengths of 488/520 nm. Labeled samples were acquired using a BD LSRFortessa FACS cytometer, and data were analyzed using FlowJo™ v10 software (Tree Star, Inc.). The compensation matrixes were designed using UltraComp eBeads™ Compensation Beads (01-222-42; Invitrogen) with specific markers.
4
Isolation and Expansion of SARS-CoV-2-Specific B Cells
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Blood samples were screened for SARS-CoV-2 RNA and for antibodies against HIV, HBV and HCV. Peripheral blood mononuclear cells (PBMCs) were isolated from heparin-treated whole blood by density gradient centrifugation (Ficoll-Paque PREMIUM, Sigma-Aldrich). After separation, PBMC were stained with Live/Dead Fixable Aqua (Invitrogen; Thermo Scientific) in 100 μL final volume diluted 1:500 at room temperature RT. After 20 min incubation cells were washed with PBS and unspecific bindings were saturated with 50 μL of 20% normal rabbit serum (Life technologies) in PBS . Following 20 min incubation at 4°C cells were washed with PBS and stained with SARS-CoV-2 S-protein labeled with Strep-Tactin®XT DY-488 (iba-lifesciences cat# 2-1562-050) for 30 min at 4°C. After incubation the following staining mix was used CD19 V421 (BD cat# 562440), IgM PerCP-Cy5.5 (BD cat# 561285), CD27 PE (BD cat# 340425), IgD-A700 (BD cat# 561302), CD3 PE-Cy7 (BioLegend cat# 300420), CD14 PE-Cy7 (BioLegend cat# 301814), CD56 PE-Cy7 (BioLegend cat# 318318) and cells were incubated at 4°C for additional 30 min. Stained MBCs were single cell-sorted with a BD FACS Aria III (BD Biosciences) into 384-well plates containing 3T3-CD40L feeder cells and were incubated with IL-2 and IL-21 for 14 days as previously described (Huang et al., 2013 (link)).
5
Flow Cytometric Analyses of Cell Phenotypes
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Flow cytometric analyses were performed using either fluorescently labeled or unlabeled mAbs followed by species-specific PE conjugates. Murine anti-human CD133 mAbs 293C3, AC133 and W6B3C1 were purchased from Miltenyi Biotec (Bergisch Gladbach, Germany). CD69-PE and CD107a-PE were from BD Pharmingen (San Diego, CA, USA), CD56-APC and CD14-PE/Cy7 from BioLegend (San Diego, CA, USA) and CD3-eFluor450 from eBioscience (San Diego, CA, USA). The goat anti-mouse PE conjugate was obtained from Dako (Glostrup, Denmark), the donkey anti-human PE conjugate was from Jackson ImmunoResearch (West Grove, PA, USA). The corresponding isotype controls were from BD Pharmingen (San Diego, CA, USA). Dead cells were excluded from analysis by 7-AAD (BioLegend; San Diego, CA, USA). Analysis was conducted using a FACS Canto II or FACS Fortessa (both BD Biosciences; Heidelberg, Germany). Specific fluorescence intensity (SFI) levels were calculated by dividing mean fluorescences obtained with a specific mAb by mean fluorescences obtained with the respective isotype control.
6
Multiparametric Characterization of Myeloid Cells
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The cells from leukapheresis, the monocytes and the DC harvested at different culture time were stained with CD14 PeCy7, CD86 APC, and HLA-DR PE (all antibodies from Biolegend, San Diego, CA, USA). Dead cells were excluded using the LIVE/DEAD™ Fixable Near-IR viability dye (Invitrogen, Carlsbad, CA, USA). Briefly, cells were washed and resuspended in FACS buffer (PBS (Bichsel, Interlakeun, Switzerland), 2% FBS (Gibco by Life Technologies, Grand Island, NY, USA), 2 mM EDTA (Life Technologies, Grand Island, NY, USA)) prior to incubation with Fc block reagent (Miltenyi Biotec, Bergisch Gladbach, Germany) for 5 min at 4 °C. The antibody mix was added before an additional 15 min incubation at 4 °C and a final cell wash in FACS buffer. Samples were acquired on a MACSQuant (Miltenyi Biotec, Bergisch Gladbach, Germany) and results were analyzed using MACSQuantify (version 2.1, Miltenyi Biotec, Bergisch Gladbach, Germany) or FlowJo (version 10.2, BD Biosciences, San Jose, CA, USA).
7
Multiparametric Immune Cell Phenotyping
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Antibodies for surface staining included CCR7 APC-Cy7 (clone G043H7; Biolegend), CCR7 APC-eFluor780 (clone 3D12; eBioscience), CD4 PE-Cy5.5 (clone S3.5; Invitrogen), CD8 BV711 (clone RPA-T8; Biolegend), CD8 Qdot 605 (clone 3B5; Invitrogen), CD14 BV510 (clone M5E2; Biolegend), CD14 Pacific Blue (clone M5E2; custom), CD14 PE-Cy5 (clone 61D3; Abcam), CD14 PE-Cy7 (clone HCD14; Biolegend), CD16 Pacific Blue (clone 3G8; custom), CD16 PE-Cy5 (clone 3G8; Biolegend), CD16 PE-Cy7 (clone 3G8; Biolegend), CD19 BV510 (clone HIB19; Biolegend), CD19 Pacific Blue (clone HIB19; custom), CD19 PE-Cy5 (clone HIB19; Biolegend), CD19 PE-Cy7 (clone HIB19; Invitrogen), CD45RO ECD (clone UCHL1; Beckman Coulter), CD45RO PE-CF594 (clone UCHL1; BD Biosciences), CD107a PE-Cy5 (clone eBioH4A3; eBioscience), CD107a PE-Cy7 (clone H4A3; Biolegend), and HLA-DR Pacific Blue (clone LN3; Invitrogen). Antibodies for intracellular staining included CD3 BV570 (clone UCHT1; Biolegend), CD3 BV650 (clone OKT3; Biolegend), CD3 Qdot 585 (clone OKT3; custom), CD3 Qdot 650 (clone S4.1; Invitrogen), Eomes Alexa 647 (WD1928; eBioscience), Eomes eFluor 660 (WD1928; eBioscience), IFN-γ Alexa 700 (clone B27; Invitrogen), Perforin BV421 (clone B-D48, Biolegend), Perforin Pacific Blue (clone B-D48; custom), Perforin PE (clone B-D48, Cell Sciences), T-bet FITC (clone 4B10; Biolegend), and T-bet PE (clone 4B10; eBioscience).
8
Multicolor Flow Cytometry of Brain Myeloid Cells
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BSLs purified from perfused brain tissue of moribund C57BL/6 mice on day 6 post-infection were stained with TCRβ-FITC, CD3ε-PE, CD4-PerCP, CD4-PerCP/Cy5.5, CD8-APC/Cy7, CD11b-APC, CD14-PE/Cy7, and F4/80-Brilliant Violet 421™ antibodies purchased from Biolegend (San Diego, CA) and fixable viability dye eFluor®506 (eBioscience, San Diego, CA). An Amnis® ImageStream®X MKII (Amnis Merck-Millipore, Seattle, WA) equipped with five lasers (355, 405, 488, 561, and 642nm) was used for acquisition. A minimum of 30,000 events were collected at 40X magnification using the INSPIRE® software (Amnis Merck-Millipore, Seattle, WA). Analysis was performed using IDEAS® 6.1 software (Amnis Merck-Millipore, Seattle, WA). Focused events were first gated using the Gradient RMS feature. Singlets were then gated by aggregate discrimination using aspect ratio and area. Analysis was then performed on gated live CD11bhighCD14+F480+TCRβ+CD3ε−CD4−CD8− cells.
9
Whole Blood Cytokine Secretion Assay
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Whole
blood stimulations were performed as described above, except that
cells were stimulated for 2 h 37 °C, 5% CO2, followed
by 4 h stimulation in the presence of Brefeldin A (BD Bioscience)
and Monensin (BD Bioscience) according to manufacturer’s recommendations
to block cytokine secretion. Subsequently, red blood cell lysis is
performed as described above and cells are stained with LIVE/DEAD
Fixable Dead Cell Stain Kit-Far Red (Invitrogen) according to manufacturer’s
recommendations. To allow intracellular cytokine staining, the cells
are treated with Cytofix/Cytoperm kit (BD Bioscience) according to
manufacturer’s recommendations. The following markers were
used to identify cell subsets: CD45-BV510 (BD Bioscience), CD3-APC-Cy7
(BD Bioscience), CD14-PeCy7 (Biolegend), and CD66b-BV421 (BD Bioscience).
Each sample divided was separately analyzed for the presence of IL-6
(IL-6-PECF594, Clone: MQ2-13A5, BD Bioscience), IL-8 (IL-8-PECF594,
clone: G265–8, BD Bioscience), or TNF-α (TNF-α-PECF594,
clone: MAB11, BD Bioscience). Flow cytometry data were collected on
a BD LSR II and analyzed using FlowJo 10.2 Software (Figure S2 ). Dead cells and CD45 negative events were excluded
from the data analysis.
blood stimulations were performed as described above, except that
cells were stimulated for 2 h 37 °C, 5% CO2, followed
by 4 h stimulation in the presence of Brefeldin A (BD Bioscience)
and Monensin (BD Bioscience) according to manufacturer’s recommendations
to block cytokine secretion. Subsequently, red blood cell lysis is
performed as described above and cells are stained with LIVE/DEAD
Fixable Dead Cell Stain Kit-Far Red (Invitrogen) according to manufacturer’s
recommendations. To allow intracellular cytokine staining, the cells
are treated with Cytofix/Cytoperm kit (BD Bioscience) according to
manufacturer’s recommendations. The following markers were
used to identify cell subsets: CD45-BV510 (BD Bioscience), CD3-APC-Cy7
(BD Bioscience), CD14-PeCy7 (Biolegend), and CD66b-BV421 (BD Bioscience).
Each sample divided was separately analyzed for the presence of IL-6
(IL-6-PECF594, Clone: MQ2-13A5, BD Bioscience), IL-8 (IL-8-PECF594,
clone: G265–8, BD Bioscience), or TNF-α (TNF-α-PECF594,
clone: MAB11, BD Bioscience). Flow cytometry data were collected on
a BD LSR II and analyzed using FlowJo 10.2 Software (
from the data analysis.
10
NK and CD3+CD56+ Cell Proliferation
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