MSC were phenotypically characterized by flow-cytometry at P2-P3 to evaluate the presence of the surface markers CD13, CD90 and CD105 and the absence of CD34, CD45 and CD80, using fluorescein isothiocyanate (FITC) or phycoerythrin (PE)-conjugated monoclonal antibodies (all from BD PharMingen, San Diego, CA). To analyze peripheral blood HD-derived monocytes and B lymphocytes, anti-CD1a allophycocyanine (APC), -CD14 FITC FITC and PE, -HLA-DR FITC, CD86 peridin chlorophyll protein-cyanine (PerCp-Cy 5.5), -CD19 APC-H7, -CD27 PE, -CD38 phycoerythrin–cyanine 7 (PE-Cy7), -IgM Alexa Fluor 647 (BD PharMingen, San Diego, CA) -conjugated monoclonal antibodies were utilized. Analysis of cell population was performed by means of direct immunofluorescence with a FACSCanto flow-cytometer (BD PharMingen); calculations were performed with the FACSDiva software (Tree Star, Inc. Ashland, OR).
Pe conjugated monoclonal antibodies
Manufactured by BD
Sourced in United States
PE)-conjugated monoclonal antibodies are laboratory reagents used for the detection and quantification of specific target molecules in biological samples. These antibodies are labeled with the fluorescent dye phycoerythrin (PE), which allows for the visualization and analysis of target cells or proteins through flow cytometry or other fluorescence-based techniques.
Automatically generated - may contain errors
Lab products found in correlation
Facsdiva software, by Tree Star (2 mentions)
Fluorescein isothiocyanate (fitc), by BD (1 mentions)
Facscanto flow cytometer, by BD (1 mentions)
Pe magnetic beads, by Miltenyi Biotec (1 mentions)
Facs star, by BD (1 mentions)
Pe conjugated mouse igg isotype antibodies, by BD (1 mentions)
Facs flow cytometer, by BD (1 mentions)
Igg1 isotype control antibody, by BD (1 mentions)
Human regulatory t cell staining kit 3, by Thermo Fisher Scientific (1 mentions)
Facscalibur flow cytometer, by BD (1 mentions)
9 protocols using pe conjugated monoclonal antibodies
1
Immunophenotyping of Mesenchymal Stromal Cells
2
Purification of Murine Hematopoietic Progenitors
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Prepared bone marrow cells from 8–12-week-old BALB/c mice, then depleted lineage positive cells by using a mixture of phycoerythrin (PE)– conjugated monoclonal antibodies directed against surface antigens (derived from blood cells including CD11b, CD5, CD8a, CD4, Gr-1, CD45R, Ter119) were purchased from BD Biosciences (San Jose, CA) and PE magnetic beads were purchased from Miltenyi Biotech (Surrey, U.K.). Purified Lin (-) cells were then further purified using a cocktail solution of supravital dyes. Principally, Lin- cells were suspended in FACS stain buffer (1XHBSS buffer) containing Rhodamine-123 and Hoechst-33342 for 15 min; labeled cells were then sorted on FACS-STAR (Becton Dickinson), the Lin-Rholow/HoechstBright(LRB) represented late stage progenitor cells and the Lin-Rholow/HoechstLow(LRH) represented more primitive progenitor cells, were highly enriched from Lin- population and used for Malat1 expression analysis.
3
Characterization of Mesenchymal Stem Cell Immunotypes
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P3 cells were digested and washed twice with PBS, and then suspended in PBS at a concentration of 5 × 105 cells/mL. Fluorescein phycoerythrin (PE) conjugated monoclonal antibodies that recognize CD105, CD90, CD73, CD34, CD45, and HLA-DR (BD Biosciences) were added to the cell suspension of UC-MSCs and PU-MSCs, while antibodies that recognize CD105, CD90, CD73, CD44, CD45, and CD31 were added to the cell suspension of AD-MSCs. The mix was incubated in the dark at 4 °C. PE conjugated mouse IgG isotype antibodies (BD Biosciences) were used as negative controls. After 30 min incubation, MSC immunotypes were determined by a FACSAria flow cytometry (FS300, Amnis) and analyzed using the FlowJo software (IDEAS 6.2.187.0). The percentage of expressed cell surface antigen was calculated for 10,000 gated-cell events.
4
Mesenchymal Stem Cell Phenotyping
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MSCs were phenotypically considered by flow cytometry at P3-P4 to assess the presence of the surface markers CD73, CD90 and the absence of CD34, CD45 and fluorescein isothiocyanate (FITC) or phycoerythrin (PE)-conjugated monoclonal antibodies (all from BD PharMingen, San Diego, CA, USA). Analysis of cell populations was achieved by direct immunofluorescence using a FACS flow cytometer (BD PharMingen). Calculations were performed with FACS Diva software (Tree Star, Inc. Ashland, OR, USA).
5
Characterizing Rabbit Stem Cells
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Rabbit ASCs and BM-MSCs were characterized by flow-cytometry. Fluorescein isothiocyanate (FITC)- or phycoerythrin (PE)-conjugated monoclonal antibodies (BD PharMingen, San Diego, CA, USA) anti-human CD29, anti-rat CD45, anti-human CD49e, anti-human CD10 and anti-rat CD90 cross-reactive against rabbit were used as described [24 (link)]. Appropriate, isotype-matched, irrelevant fluorochrome-conjugated antibodies were used as controls.
6
Quantifying PMN Activation Markers by Flow Cytometry
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Low‐affinity immunoglobulin‐Fcγ receptor IIIb (CD16b) can be found on the surface membrane of mature PMNs and can be used as a PMN identification marker. Expression of receptors on the surface of PMNs was measured by flow cytometry. The PMNs were gated according to CD16 expression and the sideward scatter profile. To study PMN activation, samples were measured for the expression of phycoerythrin (PE)‐conjugated monoclonal antibodies (mAbs) (at a final concentration of 10 μg ml−1) against CD11b and CD63 and for expression of fluorescein isothiocyanate (FITC)‐conjugated mAb against CD66b (all from BD Biosciences). Samples were incubated for 30 min on ice in the dark. Expression of cell‐surface markers was measured on the FL‐1 (Green) and FL‐2 (Red) channels and calculated as mean fluorescence intensity (MFI). Results were corrected for non‐specific binding, by subtracting the MFI values of the corresponding IgG1 isotype‐control antibodies (BD Biosciences).
7
Multicolor Flow Cytometry Profiling
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FITC-conjugated monoclonal antibodies against CD80 and HLA-DR, as well as PE-conjugated monoclonal antibodies against CD86, CD40, CD45, and HLA-ABC were purchased from BD biosciences (San Jose, CA, USA). PE-conjugated anti-HLA-G antibody was purchased from Abcam (USA). Human Regulatory T Cell Staining Kit #3 (eBiosciences, USA) was used for detecting CD4, CD25, FOXP3 positive cells (Table I).
8
Flow Cytometric Analysis of MSCs
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After three passages PB MSCs and BM MSCs were harvested and 5×105 cells were washed with FACS buffer (2% FBS, 0.1% NaN3 in PBS), incubated with 3% rat serum on ice for 30 min, and immunolabeled with 1 μg of PE-conjugated monoclonal antibodies against mouse CD14, CD34, CD44, CD105, CD117, Ly-6A/E (Sca-1) (BD); Oct-4 (Millipore); or CD29 (eBioscience) on ice for 30 min.
Fluorescent antibodies used for rabbit cells included fluorescein isothiocyanate (FITC)-conjugated anti-rabbit CD14, and phycoerythrin (PE)-conjugated anti-rabbit CD29. Analyses were performed with a FACS Calibur flow cytometer using Calibur software (Becton-Dickinson) with 10,000 events recorded for each sample.
Fluorescent antibodies used for rabbit cells included fluorescein isothiocyanate (FITC)-conjugated anti-rabbit CD14, and phycoerythrin (PE)-conjugated anti-rabbit CD29. Analyses were performed with a FACS Calibur flow cytometer using Calibur software (Becton-Dickinson) with 10,000 events recorded for each sample.
9
Mesenchymal Stem Cell Characterization
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Mesenchymal stem cells were characterized using mono-color cytofluorimetric analysis according to our previous protocol [18, (link)33] (link). To do so, we incubated 10 5 cells with 10% goat serum at a temperature of 4 °C. After 1h, the serum was withdrawn and incubation of cells was performed by phycoerythrin (PE)-conjugated monoclonal antibodies or fluorescein isothiocyanate (FITC) against human CD34, CD45, CD44, CD73, and CD90 (all from BD bioscience; cat#348057, cat#347463, cat#347943, cat#561014, cat#561970) at 4 °C for a period of 40 min. The control consisted of isotype-matched antibodies. The expression of antigens was detected using Becton-Dickinson flow cytometer and data analysis was carried out by FlowJo software.
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