H9‐derived MSCs and human bone marrow‐derived MSCs (Lonza, Walkersville, MD,
http://www.lonza.com ) were grown to confluence, harvested by using 0.25% trypsin/EDTA, and resuspended in buffer containing phosphate‐buffered saline (PBS), 2% HEPES buffer, 2% FBS, and 0.1% bovine serum albumin (BSA) as previously described 43 . Cells (1 × 106) were incubated with phycoerythrin (PE) mouse anti‐human CD90, PE mouse anti‐human CD73, fluorescein isothiocyanate (FITC) mouse anti‐human CD44, FITC mouse anti‐human CD45, FITC mouse anti‐human HLA‐ABC, PE mouse anti‐human CD29, PE mouse anti‐human CD166, PE mouse anti‐human HLA‐DR, FITC mouse anti‐human CD105, or FITC mouse anti‐human CD31 (BD Biosciences, San Jose, CA,
http://www.bdbiosciences.com ). Nonspecific fluorescence was determined by using isotype‐matched monoclonal antibodies. A total of 10,000 events were collected on a BD fluorescence‐activated cell sorting Calibur Flow Cytometer instrument by using CellQuest software (BD Biosciences). Analyses of results and corresponding graphs were generated by using FlowJo software (Tree Star, Ashland, OR,
http://www.flowjo.com ) 43 44 45 .
Fitc mouse anti human cd31
Manufactured by BD
Sourced in United States
FITC Mouse Anti-Human CD31 is a monoclonal antibody that binds to the CD31 antigen expressed on the surface of human endothelial cells. It is conjugated with the fluorescent dye FITC (Fluorescein Isothiocyanate) for detection and analysis purposes.
Automatically generated - may contain errors
Lab products found in correlation
Pe mouse anti human cd73, by BD (4 mentions)
Fitc mouse anti human cd45, by BD (3 mentions)
Fitc mouse anti human hla abc, by BD (3 mentions)
Pe mouse anti human cd90, by BD (2 mentions)
Pe mouse anti human hla dr, by BD (2 mentions)
Pe mouse anti human cd29, by BD (2 mentions)
Fitc mouse anti human cd105, by BD (2 mentions)
Fitc mouse anti human cd44, by BD (2 mentions)
Apc mouse anti human cd105, by BD (2 mentions)
Percp cy5.5 mouse anti human cd90, by BD (2 mentions)
Facscanto 2, by BD (2 mentions)
Pe mouse anti human cd166, by BD (1 mentions)
Calibur flow cytometer, by BD (1 mentions)
Cellquest software, by BD (1 mentions)
Human bone marrow derived mscs, by Lonza (1 mentions)
Fitc mouse anti human cd34, by BD (1 mentions)
Pe mouse anti human cd309, by BD (1 mentions)
Facscalibur, by BD (1 mentions)
Facsaria 2 cell sorter, by BD (1 mentions)
Af488 human alpha smooth muscle actin, by R&D Systems (1 mentions)
9 protocols using fitc mouse anti human cd31
1
Characterization of Mesenchymal Stem Cells
2
Characterization of ECFC Surface Markers
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ECFCs scraped by Corning cell lifter (Sigma, St. Louis, MO, USA) were suspended in PBS and 1 × 106 cells were stained by specific antibodies at 4 °C for 20 minutes according to the manufacturer’s instructions. The antibodies used were FITC Mouse anti-Human CD34 (BD Pharmingen, Franklin Lakes, NJ, USA), FITC Mouse anti-Human CD31 (BD Pharmingen), FITC Mouse anti-Human CD45 (BD Pharmingen), FITC Mouse anti-Human CD144 (BD Pharmingen), PE Mouse anti-Human CD309 (BD Pharmingen), FITC Mouse IgG1, κ Isotype Control (BD Pharmingen), PE Mouse IgG1, κ Isotype Control (BD Pharmingen). To obtain single cell suspensions for FACS, cells were passed through a 40-μm cell strainer and transferred to a 5 ml polystyrene round-bottom Falcon tube (BD Pharmingen). ECFC surface antigens were detected by BD FACSCalibur (BD Biosciences, San Jose, CA) and the percentages of cell expressing the surface markers were calculated by FlowJo (BD Biosciences).
3
Flow Cytometric Immunophenotyping of iMPCs
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iMPCs were trypsinized, washed and incubated with the following antibodies: FITC Mouse Anti-Human CD31, CD34, CD45, CD73, and CD90 (BD Biosciences, Franklin Lakes, NJ). Cells were then analyzed by flow cytometry (BD FACS AriaTM II cell sorter; BD Biosciences) to assess the expression of these cell surface epitopes.
4
Phenotypic Analysis of Endothelial Cells
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Cells (PAECs and HUVECs) were washed in PBS twice, dissociated with trypsin (Gibco, #25300054), and centrifuged at 400 g for 4 min. The cell pellets were washed with PBS + 5% FBS twice and resuspended in 240 µL PBS + 5% FBS. PAECs were stained with ACTA2 (AF488 Human Alpha-Smooth muscle actin, R&D, Cat#IC1420G, 1:100 dilution), CD31 (Porcine CD31/PECAM1 AF700, R&D, #FAB33871N-100UG, 1:100 dilution), and CD45 (BV421 Mouse Anti-Human CD45, BD Bioscience, #563879, 1:100 dilution). HUVECs were stained with CD31 (FITC Mouse Anti-Human CD31 (BD Bioscience, #555445), 1:100 dilution) and ACTA2 (AF488 Human Alpha-Smooth muscle actin, R&D, Cat#IC1420G, 1:100 dilution) separately. Cells were incubated on ice in the dark for 30 min. Before FC analysis, the stained cells were washed twice with pre-cooled PBS + 5% FBS. At the final wash, the cells were resuspended in 200 µL PBS + 5% FBS. Prior to this study, we compared the pig and human ACTA2 amino acid sequences, which are identical. Antibody validation of anti-ACTA2 was also validated by staining human mesenchymal stem cells. Flow cytometry was performed with the NovoCyte Quanteon analyzer (Acea bioscience, Inc, US) provided by the FACS Core Facility, Aarhus University.
5
Phenotypic Characterization of NHAC-kn Cells
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NHAC-kn cells (passage 5) were grown to near confluence, harvested by 0.25% trypsin/EDTA, washed with PBS and re-suspended in staining solution consisting of 2% FBS and 2% HEPES in PBS. Cell suspensions (1 × 106 cells) were mixed with PE mouse anti-human CD90 (BD Pharmingen), PE mouse anti-human CD73 (BD Pharmingen), PE mouse anti-human CD29 (BD Pharmingen), PE mouse anti-human HLA-DR (BD Pharmingen), FITC mouse anti-human CD44 (BD Pharmingen), FITC mouse anti-human HLA-ABC (BD Pharmingen), FITC mouse anti-human CD105 (BD Pharmingen), FITC mouse anti-human CD45 (BD Pharmingen), and FITC mouse anti-human CD31 (BD Pharmingen).28 (link) Nonspecific fluorescence was determined by incubation of cell aliquots with isotype-matched monoclonal antibodies (IgG1-PE and IgG2b-FITC). Samples were run on a Becton–Dickinson LSR II Flow Cytometer (BD Biosciences) instrument using FACS Diva software (Becton Dickinson). For each analysis, a minimum of 10,000 cells was assayed. Data was analyzed using FloJo Software (Tree Star, Inc.).
6
Characterization of Mesenchymal Stem Cells
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MSCs were immunophenotypically characterised by flow cytometry on a BD FACS Canto II (BD Biosciences). As the negative control we used 106 unstained MSCs. Test samples contained 106 MSCs with PE Mouse Anti-Human CD73, APC Mouse Anti-Human CD105, PerCP-Cy5.5 Mouse Anti-Human CD90, FITC Mouse Anti-Human CD31 and PerCP-Cy7 Mouse Anti-Human CD45 (all BD Biosciences). In addition, FACS analysis was used to determine the effect of NMP on the survival of MSCs during perfusion without a kidney with pre-labelled MSCs. For this purpose, hourly perfusate samples were taken. The MSCs could be identified by their fluorescence emission at two different wavelengths as a result of the PKH26 and Q-tracker 655 pre-labelling.
Flow cytometry was also performed on perfusate samples and enzymatically disrupted kidney biopsies from the experiments with pre-labelled MSCs with a kidney in the perfusion circuit. Of each sample, 1 million counts were obtained and analysed through flow cytometry.
Flow cytometry was also performed on perfusate samples and enzymatically disrupted kidney biopsies from the experiments with pre-labelled MSCs with a kidney in the perfusion circuit. Of each sample, 1 million counts were obtained and analysed through flow cytometry.
7
Immunophenotyping of Mesenchymal Stem Cells
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The immunophenotype characterization of the MSCs was conducted using a previously described protocol [1 (link)]. Conjugated monoclonal antibodies against PE-Cy™5 mouse anti-human CD90, PE-Cy™7 mouse anti-human CD73, APC mouse anti-human CD13, FITC mouse anti-human HLA-ABC, APC mouse anti-human CD34, FITC mouse anti-human CD31, PE mouse anti-human CD45, PE mouse anti-human CD14 (BD Biosciences, San Diego, CA, USA), eFluor™450 mouse anti-human CD105, PE mouse anti-human CD29 (eBioscience, San Diego, CA, USA), PE/Cyanine7 mouse anti-human HLA-DR and PE/Cyanine7 mouse anti-human CD10 (BioLegend, San Diego, CA, USA) were used.
8
Multiplex Flow Cytometry for MSC Characterization
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To confirm that the cells cultured were indeed MSCs, we performed flow cytometric analysis on the cells that were used during the experiments on a BD FACS Canto II (BD Biosciences Nederland BV, Vianen, The Netherlands). MSCs were stained with FITC Mouse Anti-Human CD31, PE Mouse Anti-Human CD73, PerCP-Cy5.5 Mouse Anti-Human CD90, and APC Mouse Anti-Human CD105 (all BD Biosciences Nederland BV). As negative control, unstained MSCs were used.
9
Flow Cytometry Analysis of Cell Subpopulations
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Flow cytometry scans were performed to evaluate the evolution of each cell subpopulation with time. To do this, at each selected time points (2 and 7 days), cells in 5 replicates per group were trypsinized, pooled, and subsequently blocked for 30 min with BSA 1% in DPBS. As suggested by the supplier, a minimum of 1 x 10 6 cells per group were incubated in a 100-µl test sample for 30 min at 4°C with FITC mouse anti-human CD31 (1:5, BD Bioscience, 560984) and APC mouse anti-human CD90 (1:20, BD Bioscience, 561971) diluted in PBSA. Finally, cells were rinsed twice, re-suspended with DPBS and scanned in a High Speed Cell Sorter MoFlo flow cytometer (Beckman-Coulter, CA, USA).
1:300 propidium iodide (1 mg/mL in water, Sigma-Aldrich) was added to the cell suspension to discard dead cells from the analyses.
1:300 propidium iodide (1 mg/mL in water, Sigma-Aldrich) was added to the cell suspension to discard dead cells from the analyses.
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