Cd14 apc cy7, by BD - Product details

1

Comprehensive B and T Cell Profiling

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Donor PBMCs was stained with live-dead marker (1:1000; Invitrogen, LIVE/DEAD™ Fixable Near-IR Dead Cell Stain Kit) to gate out the dead cells for 20 min in 4 °C. After washing with FACS buffer, the cells were stained with respective B, T cell marker panel antibodies diluted in FACS buffer (1xPBS, 2% FBS, 0.1% sodium azide) at 4 °C for 30 min. B cell panel: CD19-BV510 (1:40; Biolegend, Cat. No. 363020), CD14-APC-Cy7 (1:50; BD, Cat. No. 557831), CD3-APC-Cy7 (1:125; Biolegend, Cat. No. 317342), CD10-PE-Dazzle594 (1:50; Biolegend, Cat. No. 312228), CD21-APC (1:50; Biolegend, Cat. No. 354906), CD27-BV650 (1:40; Biolegend, Cat. No. 302828), CD38-PE-Cy7 (1:40; Biolegend, Cat. No. 356608), PD-1-BV711 (1:20; Biolegend, Cat. No. 329928), IgD-BUV 737 (1:40; BD, Cat. No. 612798), IgG-PerCP (1:40; BD), IgA-PE (1:40; Miltenyi, Cat. No. 130-114-002). T cell panel: CD14-APC-Cy7 (1:50; BD, Cat. No. 557831), CD19-APC-Cy7 (1:50; BD, Cat. No. 557791), CD3-BUV395 (1:40; BD, Cat. No. 563546), CD4-BUV496 (1:40; BD, Cat. No. 612936), CD8-PE (1:40; BD, Cat. No. 555367), CXCR5-PE-Cy7 (1:100; BD, Cat. No. 624052), CD25-BV421 (1:40; BD, Cat. No. 562442), CD127-FITC (1:20; BD, Cat. No. 557938). The samples were then washed, resuspended in FACS buffer and analysed on BD LSRFortessa flow cytometer (Data availability—Figshare 10.6084/m9.figshare.23549964).

Krishnananthasivam S., Li H., Bouzeyen R., Shunmuganathan B., Purushotorman K., Liao X., Du F., Friis C.G., Crawshay-Williams F., Boon L.H., Xinlei Q., Chan C.E., Sobota R., Kozma M., Barcelli V., Wang G., Huang H., Floto A., Bifani P., Javid B, & MacAry P.A. (2023). An anti-LpqH human monoclonal antibody from an asymptomatic individual mediates protection against Mycobacterium tuberculosis. NPJ Vaccines, 8, 127.

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Comprehensive B and T Cell Profiling

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Donor PBMCs was stained with live-dead marker (1:1000; Invitrogen, LIVE/DEAD™ Fixable Near-IR Dead Cell Stain Kit) to gate out the dead cells for 20 min in 4 °C. After washing with FACS buffer, the cells were stained with respective B, T cell marker panel antibodies diluted in FACS buffer (1xPBS, 2% FBS, 0.1% sodium azide) at 4 °C for 30 min. B cell panel: CD19-BV510 (1:40; Biolegend, Cat. No. 363020), CD14-APC-Cy7 (1:50; BD, Cat. No. 557831), CD3-APC-Cy7 (1:125; Biolegend, Cat. No. 317342), CD10-PE-Dazzle594 (1:50; Biolegend, Cat. No. 312228), CD21-APC (1:50; Biolegend, Cat. No. 354906), CD27-BV650 (1:40; Biolegend, Cat. No. 302828), CD38-PE-Cy7 (1:40; Biolegend, Cat. No. 356608), PD-1-BV711 (1:20; Biolegend, Cat. No. 329928), IgD-BUV 737 (1:40; BD, Cat. No. 612798), IgG-PerCP (1:40; BD), IgA-PE (1:40; Miltenyi, Cat. No. 130-114-002). T cell panel: CD14-APC-Cy7 (1:50; BD, Cat. No. 557831), CD19-APC-Cy7 (1:50; BD, Cat. No. 557791), CD3-BUV395 (1:40; BD, Cat. No. 563546), CD4-BUV496 (1:40; BD, Cat. No. 612936), CD8-PE (1:40; BD, Cat. No. 555367), CXCR5-PE-Cy7 (1:100; BD, Cat. No. 624052), CD25-BV421 (1:40; BD, Cat. No. 562442), CD127-FITC (1:20; BD, Cat. No. 557938). The samples were then washed, resuspended in FACS buffer and analysed on BD LSRFortessa flow cytometer (Data availability—Figshare 10.6084/m9.figshare.23549964).

Krishnananthasivam S., Li H., Bouzeyen R., Shunmuganathan B., Purushotorman K., Liao X., Du F., Friis C.G., Crawshay-Williams F., Boon L.H., Xinlei Q., Chan C.E., Sobota R., Kozma M., Barcelli V., Wang G., Huang H., Floto A., Bifani P., Javid B, & MacAry P.A. (2023). An anti-LpqH human monoclonal antibody from an asymptomatic individual mediates protection against Mycobacterium tuberculosis. NPJ Vaccines, 8, 127.

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Comprehensive B and T Cell Profiling

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Donor PBMCs was stained with live-dead marker (1:1000; Invitrogen, LIVE/DEAD™ Fixable Near-IR Dead Cell Stain Kit) to gate out the dead cells for 20 min in 4 °C. After washing with FACS buffer, the cells were stained with respective B, T cell marker panel antibodies diluted in FACS buffer (1xPBS, 2% FBS, 0.1% sodium azide) at 4 °C for 30 min. B cell panel: CD19-BV510 (1:40; Biolegend, Cat. No. 363020), CD14-APC-Cy7 (1:50; BD, Cat. No. 557831), CD3-APC-Cy7 (1:125; Biolegend, Cat. No. 317342), CD10-PE-Dazzle594 (1:50; Biolegend, Cat. No. 312228), CD21-APC (1:50; Biolegend, Cat. No. 354906), CD27-BV650 (1:40; Biolegend, Cat. No. 302828), CD38-PE-Cy7 (1:40; Biolegend, Cat. No. 356608), PD-1-BV711 (1:20; Biolegend, Cat. No. 329928), IgD-BUV 737 (1:40; BD, Cat. No. 612798), IgG-PerCP (1:40; BD), IgA-PE (1:40; Miltenyi, Cat. No. 130-114-002). T cell panel: CD14-APC-Cy7 (1:50; BD, Cat. No. 557831), CD19-APC-Cy7 (1:50; BD, Cat. No. 557791), CD3-BUV395 (1:40; BD, Cat. No. 563546), CD4-BUV496 (1:40; BD, Cat. No. 612936), CD8-PE (1:40; BD, Cat. No. 555367), CXCR5-PE-Cy7 (1:100; BD, Cat. No. 624052), CD25-BV421 (1:40; BD, Cat. No. 562442), CD127-FITC (1:20; BD, Cat. No. 557938). The samples were then washed, resuspended in FACS buffer and analysed on BD LSRFortessa flow cytometer (Data availability—Figshare 10.6084/m9.figshare.23549964).

Krishnananthasivam S., Li H., Bouzeyen R., Shunmuganathan B., Purushotorman K., Liao X., Du F., Friis C.G., Crawshay-Williams F., Boon L.H., Xinlei Q., Chan C.E., Sobota R., Kozma M., Barcelli V., Wang G., Huang H., Floto A., Bifani P., Javid B, & MacAry P.A. (2023). An anti-LpqH human monoclonal antibody from an asymptomatic individual mediates protection against Mycobacterium tuberculosis. NPJ Vaccines, 8, 127.

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Monocyte-to-Dendritic Cell Differentiation Assay

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PBMCs were collected using a ficoll gradient (Ficoll-Paque Plus, GE Healthcare) and monocytes were isolated by a CD14 positive selection (Miltenyi, Bergisch Gladbach, Germany). Monocytes were split into five experimental groups: (1) Negative Control (no cytokines), (2) GM–CSF (Sanofi) + IL-4 (Cell Genix) at 1000 U/ml, 3) Recombinant IL32α (R&D Systems) at 100 ng/ml, 4) Recombinant IL32β (R&D Systems) at 100 ng/ml, 5) Recombinant IL32γ (R&D Systems) at 100 ng/ml, and cultured using Cell Genix Media to yield immature DCs at day 5. Immature DC were harvested and surface stained for flow cytometry analysis. Cell surface markers were observed on the double positive, HLA-DR and CD86 population of cells. Antibodies used included CD80 FITC (BD, Clone L307.4), Mouse IgG1 FITC (Beckman Coulter PN IM0639U), CD86 Pe-Cy7 (BD, Clone FUN-1), HLA-DR PerCpCy5.5 (BD, Clone G46-4), CD1B APC (BioLegend, Clone SN13), CD14 APC-Cy 7 (BD, Clone MφP9), CD68 BV 711 (BD, Clone Y1/82A), Mouse IgG2B BV 711 (BD, Clone 27-35), and Zombie Aqua Viability Dye BV 510 (BioLegend).

Paz H., Tsoi J., Kalbasi A., Grasso C.S., McBride W.H., Schaue D., Butterfield L.H., Maurer D.M., Ribas A., Graeber T.G, & Economou J.S. (2019). Interleukin 32 expression in human melanoma. Journal of Translational Medicine, 17, 113.

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Comprehensive Leukocyte Phenotyping Protocol

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Leukocyte phenotyping has been described in a recent publication by our group.20 (link) Peripheral blood mononuclear cells (PBMC) were isolated from human peripheral whole blood by Ficoll-Paque density gradient centrifugation, and the pellet was re-suspended with a dilution of 1×10⁶ cells/mL in Fluorescence-Activated Cell Sorting (FACS) buffer. 100μL suspension was then distributed in 4 tubes:

Tube 1: not stained, containing 100μL of the PBMC suspension.

Tube 2: for CD3 assessment: 100μL of the PBMC suspension, Monoclonal antibodies (3μL CD3 FITC - BD Bioscience 561,806 + 40μL FACS Buffer);

Tube 3: for T and B cell phenotyping; 100μL of the PBMC suspension, Monoclonal antibodies (3μL CD3 FITC + 3μL CD4 PE - BD Bioscience 555,347 + 2μL CD8 PE-Cy7 - BD Bioscience 557,746 + 3μL CD19 PE-Cy5 - BD Bioscience 555,414);

Tube 4: for monocytes (M1, M2) and natural killers (NK) phenotyping: (3μL CD3 FITC + 2μL CD56 PE-Cy7 - BD Bioscience 557,747 + 3μL CD16APC - BD Bioscience 561,248 + 2μL CD14APC-Cy7 - BD Bioscience 561,384 + 3μL HLA-Dr PerCP - BD Bioscience 347,402).

Incubation for staining was carried out at 4°C for 30 minutes and the tubes were subsequently washed with 2mL FACS buffer, spinned at 1500rpm × 5min, dechanted, and re-suspended in 500μL of FACS buffer.
The tubes were analyzed with FACScanto 2 BD USA and with BD FACSdiva v8.1.

Bacigaluppi S., Bragazzi N.L., Ivaldi F., Benvenuto F., Uccelli A, & Zona G. (2022). Systemic Inflammatory Response in Spontaneous Subarachnoid Hemorrhage from Aneurysmal Rupture versus Subarachnoid Hemorrhage of Unknown Origin. Journal of Inflammation Research, 15, 6329-6342.

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Macrophage Immunophenotyping by Flow Cytometry

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As described above, macrophages were harvested using 1% (v/v) trypsin/EDTA (Life Technologies), washed with PBS and labeled with a master mix of fluorophore‐labeled human‐specific antibodies CD163‐FITC, CD80‐PE, HLA‐DR‐PE/Cy7, CD206‐APC (all BioLegend), CD14‐APC/Cy7 (BD Biosciences), and a Live/Dead violet fixable staining kit (Molecular Probes).^[⁵⁴, ⁶¹^] Samples were measured with FACS Canto II (BD Biosciences) and analyzed using FlowJo Version 8.8.6 (TreeStar Inc.). Surface marker expression levels were normalized to the unstimulated controls (set as 1).

Zbinden A., Layland S.L., Urbanczyk M., Carvajal Berrio D.A., Marzi J., Zauner M., Hammerschmidt A., Brauchle E.M., Sudrow K., Fink S., Templin M., Liebscher S., Klein G., Deb A., Duffy G.P., Crooks G.M., Eble J.A., Mikkola H.K., Nsair A., Seifert M, & Schenke‐Layland K. (2020). Nidogen‐1 Mitigates Ischemia and Promotes Tissue Survival and Regeneration. Advanced Science, 8(4), 2002500.

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Sorting Antigen-Specific B Cells

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Antigen-specific B cells were sorted from donor PBMCs by fluorescence-activated cell sorting. Briefly, frozen cells were quickly thawed at 37°C, and washed 3× with DPBS without Ca²⁺ or Mg²⁺ (Gibco) supplemented with 1% BSA (Sigma) (DPBS-BSA) before counting. Cells were resuspended in DPBS-BSA and stained with antibodies against cell markers including viability dye (Ghost Red 780) (Tonbo Biosciences), CD14-APC-Cy7 (BD Biosciences), IgM-APC-Cy7 (BD Biosciences), CD3-FITC (BD Biosciences), CD19-BV711 (BD Biosciences), and IgG-PE-Cy5 (BD Biosciences). Additionally, fluorescently-labeled antigen-oligo conjugates were added to the stain. After staining in the dark for 20 min at room temperature, cells were washed three times with DPBS-BSA. Live, CD14⁻, IgM⁻, CD3⁻, CD19⁺, Antigen⁺-cells were sorted using a FACSAria III flow sorter (BD Biosciences) and transferred to the Vanderbilt Technologies for Advanced Genomics (VANTAGE) sequencing core at an appropriate target concentration for 10X Genomics library preparation and subsequent sequence analysis.

Pilewski K.A., Wall S., Richardson S.I., Manamela N.P., Clark K., Hermanus T., Binshtein E., Venkat R., Sautto G.A., Kramer K.J., Shiakolas A.R., Setliff I., Salas J., Mapengo R.E., Suryadevara N., Brannon J.R., Beebout C.J., Parks R., Raju N., Frumento N., Walker L.M., Fechter E.F., Qin J.S., Murji A.A., Janowska K., Thakur B., Lindenberger J., May A.J., Huang X., Sammour S., Acharya P., Carnahan R.H., Ross T.M., Haynes B.F., Hadjifrangiskou M., Crowe JE J.r., Bailey J.R., Kalams S., Morris L, & Georgiev I.S. (2023). Functional HIV-1/HCV cross-reactive antibodies isolated from a chronically co-infected donor. Cell reports, 42(2), 112044.

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8

Quantifying ASC-speck formation in sepsis

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Intracellular ASC-speck formation in vivo was determined by flow cytometry on fresh whole blood samples according to previous studies (30 (link)–32 (link)). The gating strategy is shown in Supplementary Figure 2. The following antibodies were used: CD3-PerCP, CD19-PerCP, CD56-PerCP, CD66b-PerCP, ASC-PE, and HLA-DR PE-Cy7 are from BioLegend (San Diego, CA); CD14 APC-Cy7 and CD16 Krome Orange are from BD Bioscience (Heidelberg, Germany) and Beckman Coulter (Krefeld, Germany), respectively. Samples of sepsis patients and healthy controls were processed in exactly the same way. In brief, 300 µl of fresh whole blood was incubated with the antibodies (CD3, CD19, CD56, CD66b, HLA-DR, CD14, and CD16) for 15 min at room temperature (RT) in the dark. Erythrocytes were lysed using FACS lysing solution (BD Biosciences) and leukocytes were fixed simultaneously. Cells were washed and permeabilized using 0.1% saponin followed by incubation with anti-ASC monoclonal antibody for 40 min at 4°C in the dark. After staining, cells were washed and flow cytometry was performed on a Gallios Flow Cytometer (Beckman Coulter). Counting beads (Molecular Probes Invitrogen, Paisley, UK) were added into each sample before acquisition to study absolute numbers of different leukocytes subsets. Flowjo software (Version 10.0.7 for windows) was used for flow cytometric analysis.

Cui J., Oehrl S., Ahmad F., Brenner T., Uhle F., Nusshag C., Rupp C., Funck F., Meisel S., Weigand M.A., Morath C, & Schäkel K. (2021). Detection of In Vivo Inflammasome Activation for Predicting Sepsis Mortality. Frontiers in Immunology, 11, 613745.

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Characterization of Extracellular Vesicles

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To characterize EVs, PFP samples were stained and acquired as previously described¹¹ using 14 different fluorochrome-conjugated antibodies in three separate panels, including CD235a-FITC, CD62P-APC, CD3-PerCP/Cy5.5, CD19-Alexa/700, CD28-FITC, CD16-V450, CD62L-APC, CD11b-PE/Cy7, CD66-PE (Biolegend), CD15-FITC (ExAlpha), CD152-APC, CD14-APC/Cy7, CD108a-PE, and CD41a-PerCP/Cy5.5 (BD Biosciences). Samples were washed with a 0.22 μm centrifugal filter (Millipore) at 500g for 5 minutes. EVs were then harvested from the top of the filter after washing, and data were acquired by an LSR II flow cytometer (BD Biosciences). Analysis of data was performed using FlowJo 7.6.5 software (Tree Star).

Norris P.J., Schechtman K., Inglis H.C., Adelman A., Heitman J.W., Vilardi R., Shah A., Roubinian N.H., Danesh A., Guiltinan A.M., Keating S.M., Lacroix J., Cohen M.J, & Spinella P.C. (2019). Influence of blood storage age on immune and coagulation parameters in critically ill transfused patients. Transfusion, 59(4), 1223-1232.

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Myeloid Cell Phenotyping with DCFDA

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The viably cryopreserved PBMC were thawed and stained with LIVE/DEAD fixable violet Dead Stain Kit (Thermo Fisher Scientific) for 20 minutes followed by staining with a myeloid panel of antibodies comprising CD14-APC-Cy7 (564123), CD33-PE-Cy7 (333952), CD16-BV605 (563172), HLA-DR-BV786 (564041), all from BD Biosciences, for 30 minutes at 4°C. Cells were then washed and stained with H2-DCFDA (D399, Molecular Probes) in serum-free Iscoves’ Modified Dulbecco's Medium (IMDM; 36531, Thermo Fisher Scientific) for 30 minutes at 37°C, washed and acquired on a five laser BD LSR Fortessa and analyzed with FlowJo.

Grauers Wiktorin H., Aydin E., Kiffin R., Vilhav C., Bourghardt Fagman J., Kaya M., Paul S., Westman B., Bratlie S.O., Naredi P., Hellstrand K, & Martner A. (2024). Impact of Surgery-Induced Myeloid-derived Suppressor Cells and the NOX2/ROS Axis on Postoperative Survival in Human Pancreatic Cancer. Cancer Research Communications, 4(4), 1135-1149.

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Cd14 apc cy7

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Close spelling variants of this product

32 protocols using cd14 apc cy7

Comprehensive B and T Cell Profiling

Comprehensive B and T Cell Profiling

Comprehensive B and T Cell Profiling

Monocyte-to-Dendritic Cell Differentiation Assay

Comprehensive Leukocyte Phenotyping Protocol

Macrophage Immunophenotyping by Flow Cytometry

Sorting Antigen-Specific B Cells

Quantifying ASC-speck formation in sepsis

Characterization of Extracellular Vesicles

Myeloid Cell Phenotyping with DCFDA

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