The cellular differentiation analysis was performed using flow cytometry. To analyze the cell surface antigen expressions, the cells after third passage was used. MSCs were incubated with antibodies for rat CD90 FITC (BD Biosciences, San Diego, CA, USA), CD 29 FITC (BD Biosciences, San Diego, CA, USA), CD106 PE (BD Biosciences, San Diego, CA, USA), CD54 PE (BD Biosciences, San Diego, CA, USA) at room temperature in the dark. Control antibodies were FITC Rat IgG2a, K isotype controls and IgG1 PE isotype controls (BD Biosciences, San Diego, CA, USA). Negative markers were CD3 PE (BD Biosciences, San Diego, CA, USA), CD4 APC (BD Biosciences, San Diego, CA, USA), CD25 FITC (BD Biosciences, San Diego, CA, USA), CD45 FITC (BD Biosciences, San Diego, CA, USA), CD8B FITC (BD Biosciences, San Diego, CA, USA).
Cd54 pe
Manufactured by BD
Sourced in United States
The CD54-PE is a laboratory instrument used for cellular analysis. It detects the expression of the CD54 surface antigen, also known as Intercellular Adhesion Molecule 1 (ICAM-1), on cells. The CD54-PE utilizes fluorescent labeling with the PE (Phycoerythrin) dye to enable the identification and quantification of CD54-positive cells.
Automatically generated - may contain errors
Lab products found in correlation
Cd45 fitc, by BD (4 mentions)
Cd44 fitc, by BD (3 mentions)
Cd49e pe, by BD (3 mentions)
Cd106 fitc, by BD (3 mentions)
Anti human cd133 apc, by BD (2 mentions)
Cd54 percp cy5, by BD (2 mentions)
Cd45 bv510, by BD (2 mentions)
Cd14 pe, by BD (2 mentions)
Hla dr fitc, by BD (2 mentions)
Cd11a fitc, by BD (2 mentions)
Cd163 pe, by BD (2 mentions)
Facscalibur, by BD (2 mentions)
Isotype control, by BD (2 mentions)
Cd29 pe, by BD (2 mentions)
Cd49a pe, by BD (2 mentions)
Cd49b pe, by BD (2 mentions)
Cd49c pe, by BD (2 mentions)
Cd49d fitc, by BD (2 mentions)
Cd49f pe, by BD (2 mentions)
Cd51 61 pe, by BD (2 mentions)
13 protocols using cd54 pe
1
Mesenchymal Stem Cell Surface Antigen Profiling
2
Characterizing CTCs in Colorectal Cancer
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Peripheral blood samples were obtained from CRC patients attending our department and an informed consent was obtained from all the individuals. Peripheral blood samples were collected and prepared as per the protocol described in our previous report 19 . In detail, CTCs from cell suspensions were characterized by multiparameter flow cytometry. The antibodies used in this study included anti‐human CD133‐APC, CD44‐FITC, CD54‐PErcp‐cy5.5, CD54‐PE, and CD45‐BV510 (all antibodies were purchased from BD Biosciences, San Diego, CA, USA). DAPI was used to identify and sort the dead cells. The remaining steps were the same as the protocol described in our previous report 19 . The absolute CTCs or antibody‐positive cell numbers were derived from the absolute number of white blood cells provided by the hematological analyzer, and the percentage of CTCs or antibody‐positive cells was determined by flow cytometry, using the following formula: percentage of cells × white blood cells count/100.
3
Characterization of Monocyte/Macrophage Phenotypes
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Cell surface markers were analyzed using specific florescence conjugated and non-conjugated antibodies. Non-specific florescence was attuned using appropriate Isotype controls. For monocyte and macrophage specific cell surface antigen expression, mouse anti-human- CD68 FITC, CD14 PE, CD11b APC, HLA-ABC FITC, HLA-DR FITC, CD80 FITC, CD86 PE, CD50 FITC, CD54 PE, CD163 PE, CD205 PE and CD206 APC antibody conjugates from BD Pharmingen; CX3CR1 FITC from R&D Systems were used. To prevent non-specific binding of antibodies to macrophage/monocyte lineage cells, cells were pre-incubated with buffer containing 2 mM EDTA, 0.5% FBS. DPBS and 10 μl of FcR blocker (Miltenyi Biotec) for 15 min at 4 °C. Cells were then washed with DPBS and stained in antibody containing FACS buffer (DPBS containing 1% FBS and 0.01% sodium azide) on ice for 1 hour. For indirect staining, cells were further washed and treated with appropriate secondary antibody for 30 minutes on ice. Stained cells were fixed with 1% Para Formaldehyde and stored at 4 °C till further analysis. Cells were acquired on a BD FACS CALIBUR and the data was analyzed using the Cell Quest Pro software. % positivity was calculated for each surface antigen after gating with respect to relevant isotype control antibody.
4
Multiparameter Flow Cytometry of CTCs
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CTCs from cells suspension were characterized by multiparameter flow cytometry. The antibodies used in this study include: anti-human CD133-APC, CD44-FITC, CD44-APC-Cy7, CD54-PErcp-cy5.5, CD54-PE, CD24-PE/Cy7, CD10-PECF594, CD26-PE, CD166-Percp-cy5.5, CD45-BV510, CD58-PE, CD66-PE, CD71-PE, CD117-PE, EPCAM-Percp-cy5.5, and EGFR-PE (all of the above-mentioned antibodies were purchased from BD Biosciences). DAPI was used to identify the dead cells. Evaluation of nucleated cells from whole cells suspensions was carried out using a FACS Canto Flow Cytometer (BD Biosciences) and data were analyzed using BD FACS Diva software. A range of internal quality assurance procedures was employed, including daily calibration of the optical alignment and fluidic stability of the flow cytometer using the seven-color Set-up Beads (BD Biosciences). The absolute CTCs or antibody-positive cell number was derived from the absolute number of the white blood cells provided by the hematological analyzer and percentage of CTCs or antibody-positive cell as determined by flow cytometry, using the following formula: percentage of cells × white blood cells count/100.
5
Phenotyping of NK Cell Subsets
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The monoclonal antibodies CD3‐PerCP, CD56‐allophycocyanin(APC), CD158a‐FITC, CD158b‐PE, CD158e‐FITC, CD94‐FITC, CD62L‐FITC, CD54‐PE, CD11a‐FITC, CX3CR1‐FITC, CXCR4‐PE, CCR7‐PE, NKP30‐FITC, NKP46‐PE, IL13‐PE, IL10‐PE, TGF‐β‐PE and IFN‐γ‐FITC (BD Bioscience, Mountain View, CA, USA), and NKG2A‐PE (BeckmanCoulter, USA) and appropriate isotypes were used in individual 4‐colour flow cytometry assays to analyse the immunophenotype as well as the cytokine secretion of NK cells. Intracellular staining was performed using the Pharmingen Intracellular Staining Kit (BD Pharmingen, San Diego, CA, USA). The cells were incubated for 5 hours with phorbol myristate acetate (PMA) (40 ng/mL) plus ionomycin (2.5 μg/mL, all reagents from Sigma Chemical) to stimulate maximal IFN‐γ, IL‐13, TGF‐β and IL‐10 production; GolgiStop (0.7 μL/mL) was added to the sample during the last 4 hours to trap the protein in the cytoplasm. NK1, NK2, NK3 and NKr cells were identified as CD3−CD56+IFN‐γ+, CD3−CD56+IL‐13+,CD3−CD56+ TGF‐β+ and CD3−CD56+IL‐10+, respectively. The dose of NK1, NK2, NK3 and NKr cells was classified as the absolute number of NK1, NK2, NK3 and NKr cells infused in GBM and GPB (cells/kg). Data were analysed using a FACSCaliber 4‐colour flow cytometer (BD Biosciences) and FlowJo 7.6.1 software (Tree Star Inc, CA, USA).
6
Enavatuzumab Modulates Immune Responses
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PBMCs from healthy human donors were added to 24-well plates, either alone or into wells that contained SN12C cells that had been plated 24 hrs previously. The cultures were incubated with enavatuzumab or a control antibody (10 μg/mL) for 24 hrs, after which the immune cells were removed to measure activation markers by flow cytometry. Immune cells were stained for monocytes and nature killer (NK) cells with fluorochrome-conjugated antibodies purchased from BD Biosciences: CD3-FITC, CD54-PE, CD16-PerCP Cy5.5, CD14-PE Cy7, CD56-APC, and CD69-APC Cy7. In some experiments, tumor cell cytotoxicity was assessed after 24 hr culture by measuring the level of cytokeratin18 in the supernatant by M65® ELISA (Peviva, Bromma, Sweden), according to the manufacturer's instructions.
7
Phenotypic and Functional Characterization of pMSCs
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5 × 105 pMSCs per tube were phenotypically characterized by flow cytometry (FACSCalibur, BD Biosciences, Franklin Lakes, NJ, USA), using the following antibodies CD105-PerCP, CD54-PE, CD44-FITC, CD49e-PE, CD166-PE, CD13-APC, HLA-ABC-PE, CD45-FITC, CD14-PE, CD51,61-FITC, CD106-FITC, CD34-PerCP, CD31-FITC, and HLA-DR-FITC (Pharmigen, BD Biosciences, Franklin Lakes, NJ, USA). Ten thousand events were recorded for each sample and data was analyzed using CellQuest software (Becton Dickinson, Franklin Lakes, NJ, USA). In addition, pMSCs were functionally characterized by multipotent differentiation in adipocytes and osteocytes, as previously described [37 (link)].
8
Flow Cytometry Analysis of DC Phenotypes
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Human DC phenotypes were assessed via flow cytometry (Becton Dickinson, LSRFortessa). Cells were incubated with fluorochrome-conjugated monoclonal antibodies (CD40-PE-Cy7, CD86-BV510, HLA-DR-APC, CD83-APC-Cy7, CD274-APC, and CD54-PE; BD PharMingen) for 30 min on ice, washed, and analyzed using FlowJo software as previously described [44 ].
9
Flow Cytometric Analysis of Dendritic Cells
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Isolated CD103+ DCs (1 × 106) were subjected to flow cytometric analysis. Mouse-anti-rat CD54-Alexa Fluor®488 (R&D Systems), CD86-phycoerythrin (PE)-Vio770 (Miltenyi Biotec), CD80-PE (eBioscience, San Diego, CA, USA), CD11b/c-allophycocyanin (APC; Biolegend, San Diego, CA, USA), RT1B (MHC- II)-FITC (BD Biosciences, San Jose, CA, USA), CD1d-APC (eBioscience), and the respective matched isotype controls were employed.
The following markers were used for the staining of imDCs (1 × 106): mouse-anti-human CD80-FITC, CD54-PE, CD86-peridinin chlorophyll (PerCP)-Cy™5.5, MHC-II-APC, CD1a-FITC, CD83-PE, CD11c-PerCP-Cy™5.5, and the respective matched isotype controls (BD Biosciences). All samples were washed, resuspended in 2% PFA, and subjected to flow cytometric analysis (Accuri C6; BD Biosciences).
The following markers were used for the staining of imDCs (1 × 106): mouse-anti-human CD80-FITC, CD54-PE, CD86-peridinin chlorophyll (PerCP)-Cy™5.5, MHC-II-APC, CD1a-FITC, CD83-PE, CD11c-PerCP-Cy™5.5, and the respective matched isotype controls (BD Biosciences). All samples were washed, resuspended in 2% PFA, and subjected to flow cytometric analysis (Accuri C6; BD Biosciences).
10
Phenotypic Characterization of CD34+ Cells
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Totally, 5 × 10 5 CD34+ cells were collected on days 0, 3, 7, 10, and 14. After washing twice with PBS, the cells were labeled using the following monoclonal antibodies: mouse antihuman CD34-FITC, CD42b-PE, CD41a-PE, CD61-FITC, CD49d-PE, CD49e-FITC, CD11a-FITC, CD54-PE, and mouse IgG 1 -FITC, IgG 2 -PE (BD, Pharmingen, USA). Cells in PBS at a volume of 100 with 10 μL of monoclonal antibody were incubated at 25°C for 30 min. After washing with PBS, the cells were resuspended in 100 μL of PBS and detected by FCM (Guava easyCyte 8HT, EMD Millipore, Billerica, MA, USA), and data were analyzed by Guava InCyte software (Version 2.8, EMD Millipore, Billerica, MA, USA).
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