Cd14 apc

Manufactured by BD

Sourced in United States, Germany

CD14-APC is a fluorescently-labeled monoclonal antibody that binds to the CD14 cell surface antigen. CD14 is a glycosylphosphatidylinositol-anchored protein that serves as a receptor for lipopolysaccharide (LPS) and is expressed on the surface of monocytes, macrophages, and other myeloid cells.

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APC anti–human CD14 Anti-CD14-APC-Cy7 (clone MφP-9; Annexin V-FITC and CD14 APC CD14 APC-Cy7 (MoP9, Mouse anti-human IgG2b CD14 –APC APC-H7-conjugated anti-CD14 Anti-CD14 APC-H7 Anti-CD14-APC-Cy7 APC mouse anti-human CD14 APC anti-human CD14 antibody

49 protocols using cd14 apc

Monocyte-Platelet Aggregation and Platelet Activation Measurement

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MPA were identified in citrate anticoagulated blood as previously described [8 (link)]. Briefly, whole blood was fixed with 1% formaldehyde (Sigma) 15 min after blood collection. Fixed whole blood was stained with 5 μL CD61-FITC (Dako) to identify platelets and 5 μL CD14-APC (BD Biosciences) to identify monocytes. After lysis of red blood cells, monocytes were collected based on side-scatter properties and positive staining for CD14 using an Accuri C6 flow cytometer (BD Biosciences). MPA were identified as having a positive stain for CD14 and CD61, and were recorded as a percent of 2000 total monocytes collected.
Platelet activation was determined by platelet surface expression of P-selectin, and CD40 with whole blood flow cytometry, as previously described [8 (link)]. Briefly, P-selectin expression was determined with a FITC-conjugated anti-CD62P antibody (BD Biosciences), and CD40 expression was determined with a FITC-conjugated anti-CD40 antibody (BD Biosciences). Gates were established to include platelets with and without aggregation to monocytes. Monocytes were identified by staining with CD14-APC (BD Biosciences), and platelets were identified by staining with CD42-PE (BD Biosciences). The expression of platelet activation markers were assessed individually

Barrett T.J., Schlegel M., Zhou F., Gorenchtein M., Bolstorff J., Moore K.J., Fisher E.A, & Berger J.S. (2019). Platelet regulation of myeloid suppressor of cytokine signaling 3 accelerates atherosclerosis. Science translational medicine, 11(517), eaax0481.

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Phenotypic Analysis of RA and OA Synovium Cells

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Flow cytometric analyses of synovium-derived cells from RA and OA were performed with digested cells before plating (day 0) and expanded cells cultured for 14 days. The cells from three donors were harvested using a cell dissociation buffer. Cells were suspended in HBSS at a density of 5 × 10⁵ cells/mL and stained for 30 minutes on ice with the antibodies CD11b-PE, CD11c-PE-Cy7, CD14-APC, CD31-FITC, CD44-APC-H7, CD45-FITC, CD73-BV421, CD90-PE, CD105-PerCP-Cy5.5, CD206-FITC, and HLA-DR-APC (Becton, Dickinson and Company; BD, Franklin Lakes, NJ, USA). Flow cytometric analysis of cell surface antigens was performed by a triple-laser FACS Verse™ system (BD).

Kohno Y., Mizuno M., Ozeki N., Katano H., Komori K., Fujii S., Otabe K., Horie M., Koga H., Tsuji K., Matsumoto M., Kaneko H., Takazawa Y., Muneta T, & Sekiya I. (2017). Yields and chondrogenic potential of primary synovial mesenchymal stem cells are comparable between rheumatoid arthritis and osteoarthritis patients. Stem Cell Research & Therapy, 8, 115.

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Flow Cytometry Analysis of Immune Cells in Ischemic Stroke

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Peripheral blood samples were collected from healthy control subjects and ischemic stroke subjects at the time of admission (<72 h after onset). Blood samples were stored in ice-cold tubes containing EDTA prior to flow cytometry analysis. Mononuclear cells were isolated from the whole-blood specimens and stained with fluorescent-labeled antibodies, including CD3-PEcy7, CD4-APC, CD8-FITC, CD19-APC, CD56-PerCP, CD11b-PerCP, CD11c-FITC, CD14-APC, CD16-FITC, and CD74-PE (Becton Dickinson, San Jose, CA). Data were acquired using FACS Caliber (Becton Dickinson, San Jose, CA) and analyzed with Flow Jo 7.6 software.

Yang L., Kong Y., Ren H., Li M., Wei C.J., Shi E., Jin W.N., Hao J., Vandenbark A.A, & Offner H. (2016). Upregulation of CD74 and its potential association with disease severity in subjects with ischemic stroke. Neurochemistry international, 107, 148-155.

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Comprehensive Immunophenotyping of Elutriated Monocytes and Dendritic Cells

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Analysis of expression of surface markers was performed using
fluorescent labeled antibodies (Abs) and flow cytometry. The purity of the
elutriated monocytes was assessed by flow cytometry using CD33-PE, CD15-FITC,
CD3/CD19/CD56-APC and CD45-APC-Cy7 (Becton Dickinson, Mountain View, CA, USA)
and isotype controls (Becton Dickinson). DC were analyzed after pulsing on Day
4. The analysis included the standard “DC panel” adopted in our
institution as lot release for mature DC products and other investigational
markers. The panel consisted of CD86-FITC, CD83-PE, CD14-APC, HLA-DR-FITC,
CD123-PE, CD11c-APC, CD80-FITC, CD54-APC, CCR7-APC, and CD38-FITC (Becton
Dickinson). Flow cytometry acquisition and analysis were performed with a
FACScanto flow cytometer (Becton, Dickinson and Company, Franklin Lakes, NJ USA)
according to CPS standard operating procedures. Spectral overlaps were
electronically compensated using single color controls. Quality controls were
run before each session according to internal quality control policy.

Castiello L., Sabatino M., Ren J., Terabe M., Khuu H., Wood L.V., Berzofsky J.A, & Stroncek D.F. (2017). Expression of CD14, IL-10 and tolerogenic signature in dendritic cells inversely correlate with clinical and immunological response to TARP vaccination in prostate cancer patients. Clinical cancer research : an official journal of the American Association for Cancer Research, 23(13), 3352-3364.

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Phenotyping of Immune Cells by Flow Cytometry

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Antibodies for surface markers staining of DCs and T cells were obtained from BD Biosciences, New York, USA (anti-human CD3-PE, CD3-FITC, CD8-PerCP, CD8-APC, CD56-PE, NKG2D-APC, CD4-FITC, CD4-PerCP, CD107a-FITC, CD25-APC, CD45RO-FITC, CD27-PerCPCY5.5, CD57-APC, CCR7-PE, CD14-APC, CD80-PE, CD83-APC, CD86-FITC, HLA-DR-FITC). Antibodies for intracellular proteins staining were also obtained from BD Biosciences (anti-human IFNγ-APC, TNFα-PECY7, granzyme B-FITC, FoxP3-PE). The intracellular staining was performed by fixing and permeabilizing cells with Cytofix/Cytoperm (BD Biosciences). The experiments were performed by using FACS CantoII (BD Biosciences) flow cytometer, and data were analyzed by using the Flowjo software.

Han Y., Wu Y., Yang C., Huang J., Guo Y., Liu L., Chen P., Wu D., Liu J., Li J., Zhou X, & Hou J. (2017). Dynamic and specific immune responses against multiple tumor antigens were elicited in patients with hepatocellular carcinoma after cell-based immunotherapy. Journal of Translational Medicine, 15, 64.

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Isolation and Characterization of MSCs

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MSCs were isolated from the iliac crest bone marrow of all the study subjects and purified using density gradient centrifugation as previously described [25 (link)]. After being resuspended in Dulbecco's Modified Eagle's Medium (DMEM, Gibco, USA) containing 10% heat-inactivated fetal bovine serum (FBS, Sijiqing, China), MSCs were seeded in 25 cm² flasks and cultured in an incubator with 5% CO₂ at 37°C. To remove the suspended cells, the medium was replaced 48 hours later and every 3 days thereafter. At 80–90% confluence, the adherent cells were recovered using 0.25% Trypsin containing 0.53 mM EDTA (Gibco, USA) and replated in 75 cm² flasks as passage 1. MSCs were expanded and used at passages 4 for all experiments. Surface markers of MSCs were detected using flow cytometry (FCM), which was performed on BD Influx cell sorter (BD, USA). CD14-APC, CD29-PE, CD44-FITC, CD45-APC, CD105-FITC, and HLA DR-PerCP antibodies were used (all from BD, USA).

Liu Z., Gao L., Wang P., Xie Z., Cen S., Li Y., Wu X., Wang L., Su H., Deng W., Wang S., Li D., Li J., Ouyang Y., Wu Y, & Shen H. (2017). TNF-α Induced the Enhanced Apoptosis of Mesenchymal Stem Cells in Ankylosing Spondylitis by Overexpressing TRAIL-R2. Stem Cells International, 2017, 4521324.

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Hydrogel-Mediated Complement Inhibition

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The amount of Atto488 released from the hydrogel as a model drug was determined by fluorescence measurement at λ_ex. = 485 nm/λ_em. = 535 nm against an Atto488 standard. The inhibitory effect of soluble as well as hydrogel‐released compstatin 4W9A was tested in whole blood, where the complement system was activated with 10 µg mL⁻¹ nonopsonized zymosan for 30 min at 37 °C. The supernatant of cleaved PMX53 conjugated hydrogels was exposed to isolated granulocytes in the presence of 5% zymosan‐activated serum for 20 min at 37 °C. Cell activation was determined by flow cytometry using CD11b‐VioBlue staining (Biolegend) for isolated granulocytes and additionally CD14‐APC and CD41a‐FITC (both BD Biosciences, New Jersey, USA) for whole blood samples. Leukocyte elastase and C5a concentrations were determined using the commercial ELISA kits Hycult human elastase ELISA (Hycult Biotech, Uden, Netherlands) and C5a micro ELISA (DRG, Marburg, Germany) according to the manufactures instructions.

Helmecke T., Hahn D., Matzke N., Ferdinand L., Franke L., Kühn S., Fischer G., Werner C, & Maitz M.F. (2022). Inflammation‐Controlled Anti‐Inflammatory Hydrogels. Advanced Science, 10(7), 2206412.

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Multiparameter Flow Cytometry Immunophenotyping

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100 μl of 5×10⁷/ml single cell suspension with 20 μl of fluorescently labeled mouse anti-human CD13 (CD13-PE, 560998, BD, Shanghai, China, 1：500), CD14 (CD14-APC, 561708, BD, Shanghai, China, 1：500), CD34 (CD34-PerCP, 340430, BD, Shanghai, China, 1：500), CD44 (CD44-APC, 559942, BD, Shanghai, China, 1：500), CD90 (CD90-FITC, 561969, BD, Shanghai, China, 1：500), and CD105 (CD105-PE, 560839, BD, Shanghai, China, 1：500) antibodies were added to a clean plastic tube and incubated at 4℃. The cells were then added with 2 ml PBS, mixed and centrifuged at 1500 round/min for 5 min. Next, the supernatant was discarded and 200 μl PBS was added to each tube and well mixed. The tubes were loaded on flow cytometry and tested. Data was analyzed by Cell-Quest software.

Mao Y., Ma J., Xia Y, & Xie X. (2020). The Overexpression of Epidermal Growth Factor (EGF) in HaCaT Cells Promotes the Proliferation, Migration, Invasion and Transdifferentiation to Epidermal Stem Cell Immunophenotyping of Adipose-Derived Stem Cells (ADSCs). International Journal of Stem Cells, 13(1), 93-103.

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Multicolor Cytometry for Treg and MDSC

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For Treg cells, isolated PBMCs were stained with anti-CD4-FITC (RPA-T4/555346), CD25-APC (M-A251/555434), and CD45RA-PerCP-Cy 5.5 (HI100/563429) antibodies (BD Biosciences, San Jose, CA, USA) for 45 min, and antibody stained samples were washed twice. After intracellular staining, Treg cell frequencies were analyzed by a BD FACSVerse (BD Biosciences) flow cytometer. For MDSCs, isolated PBMCs were stained with anti-CD3-BV421 (UCHT1/562426), CD19-BV421 (HIB19/562440), CD56-BV421 (NCAM16.2/562751), CD20-BV421 (2H7/562873), CD11b-BB515 (ICRF44/564517), CD15-PerCP-Cy 5.5 (HI98/560828), CD14-APC (M5E2/555399), and HLA-DR-PE (G46-6/555812) antibodies (BD Biosciences) for 45 min, washed twice, and analyzed by a BD FACSVerse (BD Biosciences) flow cytometer. For 7-AAD and propidium iodide staining, isolated PBMCs were stained with 7-AAD (Biolegend, San Diego, CA, USA) or PI (BD Biosciences) for 10 min and then analyzed on a BD FACSVerse (BD Biosciences). Gating strategies are shown in Supplementary Fig. S1. PBMC viability before MDSC analysis is shown in Supplementary Fig. S2.

Koh J., Hur J.Y., Lee K.Y., Kim M.S., Heo J.Y., Ku B.M., Sun J.M., Lee S.H., Ahn J.S., Park K, & Ahn M.J. (2020). Regulatory (FoxP3+) T cells and TGF-β predict the response to anti-PD-1 immunotherapy in patients with non-small cell lung cancer. Scientific Reports, 10, 18994.

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Phenotypic Analysis of Adipose-Derived Stem Cells

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For ASC phenotype analysis, cells were detached with non-enzymatic cell dissociation solution (ATCC Manassas, VA, USA). In the next step, ASCs were washed with FACS buffer (phosphate-buffered saline, 0.1% NaN₃, 1% FCS). Then, 5 × 10⁴ cells were suspended in 50 μL of FACS buffer and stained with antibodies against the following surface markers: CD90-FITC, CD105-PE, CD73-APC (eBioscience, San Diego, CA, USA), CD34-PE-Cy7, CD45-PE, CD19-PE, and CD14-APC (BD Pharmingen, San Diego, CA, USA). After the washing step, cells were acquired and analysed using a FACSCanto cell sorter/cytometer and Diva software. The gating strategy is shown in Figure S1 in the Supplementary Materials.

Kuca-Warnawin E., Kurowska W., Plebańczyk M., Wajda A., Kornatka A., Burakowski T., Janicka I., Syrówka P, & Skalska U. (2023). Basic Properties of Adipose-Derived Mesenchymal Stem Cells of Rheumatoid Arthritis and Osteoarthritis Patients. Pharmaceutics, 15(3), 1003.

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