The phenotype of NPCs was determined by cytofluorimetric analysis with fluorochrome‐conjugated antibodies against human CD90‐FITC (#555595) at 1:100, CD73‐PE (#550257) at 1:100, CD105‐Alexa488 (#MHCD10520) at 1:100, CD34‐PECy5 (#555823) at 1:100, CD45‐PECy7 (#557748) at 1:100, CD146‐BV605 (#7433019) at 1:100, and Tie2‐PE (#FAB3131A) at 1:50 (all from BD Bioscience, Franklin Lakes, NJ). Isotype IgG was used as a control (all from BD Biosciences). Cells in suspension were incubated for 40 minutes with each of these antibodies at 4°C in FACS buffer (PBS, 0.5% human serum albumin, 0.5 mm EDTA), and analyzed with a Cell Lab Quanta SC flow cytometer (Beckman Coulter Inc.).
Cd34 pe cy5
Manufactured by BD
Sourced in United States
CD34-PE-Cy5 is a fluorescently-labeled antibody that binds to the CD34 protein, a cell surface marker expressed on hematopoietic stem and progenitor cells. The PE-Cy5 fluorescent label allows for the identification and enumeration of CD34-positive cells in flow cytometry analysis.
Automatically generated - may contain errors
Lab products found in correlation
Cd14 pe, by BD (4 mentions)
Cd45 fitc, by BD (3 mentions)
Cd90 pe, by BD (3 mentions)
Cd31 pe, by BD (3 mentions)
Cd73 pe, by BD (2 mentions)
Cd73 fitc, by BD (2 mentions)
Cd146 pe, by BD (2 mentions)
Cd54 pecy5, by BD (2 mentions)
Cd105 fitc, by BD (2 mentions)
Cd44 pe, by BD (2 mentions)
Cd166 pe, by BD (2 mentions)
Hla dr fitc, by BD (2 mentions)
Cd45 pe cy7, by BD (1 mentions)
Isotype igg, by BD (1 mentions)
Cell lab quanta sc, by Beckman Coulter (1 mentions)
Cd3 qdot605, by Thermo Fisher Scientific (1 mentions)
Cd90 apc, by BD (1 mentions)
Cd25 apc cy7, by BD (1 mentions)
Cd4 qdot655, by Thermo Fisher Scientific (1 mentions)
Cd106 pe cy5, by BD (1 mentions)
10 protocols using cd34 pe cy5
1
Characterizing NPC Phenotype by Flow Cytometry
2
Phenotypic Characterization of T-cell Subsets
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MSCs were analyzed using the following antibodies: CD73-PE, CD105-PE, CD14-PE, CD45-FITC, CD90-APC (BD Biosciences), CD106-Pe-Cy5, CD34-Pe-Cy5 (BD Biosciences), HLA-DR-Qdot-605 (Invitrogen), mouse mAb specific to FAP (eBioscience) and PE-conjugated anti-mouse (BD Biosciences). Isotype-matched, fluorochrome-conjugated, mAb were used as negative controls.
Cultured SCS were stained, data acquired and analyzed as previously described using the following antibodies [11] (link); Surface: CD3-Qdot-605, CD4-Qdot-655 (Invitrogen), CXCR5-Alexa-488, CD25-APC-Cy7, PD-1-Brilliant Violet-421; Intracellular: Bcl-6-PE-CF594, active caspase-3-PE (BD Biosciences), and FoxP3-PE-Cy7 (eBioscience).
FL T-cell subsets were flow cytometrically sorted using the following markers; T-cell: DAPI−CD3+CD4+CD19−; TFH: Propidium iodide−CD3+CD4+CXCR5+PD-1+CD25−. Following the sort, an aliquot of TFH were permeabilized and stained with Bcl-6 and FoxP3 to confirm the TFH phenotype. TFH cells were defined as CD3+CD4+CXCR5+PD-1+CD25−Bcl-6+; TFR were defined as CD3+CD4+CXCR5+PD-1+CD25+Bcl-6+FoxP3+ and Tregs were defined as CD3+CD4+CD25+FoxP3+.
Cultured SCS were stained, data acquired and analyzed as previously described using the following antibodies [11] (link); Surface: CD3-Qdot-605, CD4-Qdot-655 (Invitrogen), CXCR5-Alexa-488, CD25-APC-Cy7, PD-1-Brilliant Violet-421; Intracellular: Bcl-6-PE-CF594, active caspase-3-PE (BD Biosciences), and FoxP3-PE-Cy7 (eBioscience).
FL T-cell subsets were flow cytometrically sorted using the following markers; T-cell: DAPI−CD3+CD4+CD19−; TFH: Propidium iodide−CD3+CD4+CXCR5+PD-1+CD25−. Following the sort, an aliquot of TFH were permeabilized and stained with Bcl-6 and FoxP3 to confirm the TFH phenotype. TFH cells were defined as CD3+CD4+CXCR5+PD-1+CD25−Bcl-6+; TFR were defined as CD3+CD4+CXCR5+PD-1+CD25+Bcl-6+FoxP3+ and Tregs were defined as CD3+CD4+CD25+FoxP3+.
3
Flow Cytometry Immunostaining Analysis
4
Immunophenotyping of Cultured Cells
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After cell detachment using a 0.125% trypsin solution, cells were washed with PBS and resuspended in PBS containing 2% FBS. Cell concentration and viability were monitored using Trypan blue in a Neubauer haemocytometer. The following monoclonal antibodies were used as indicated by the manufacturer (BD Pharmingen): CD90-PE (BD, #555596), CD73-FITC (BD, #561254), CD105-FITC (BD,#561443), CD45-FITC (BD,#347463), CD14-PE (BD,#555398), CD34-PEcy5 (BD,#561819), CD31-PE (BD,#555446), IgG-FITC (BD,#555786), HLA-DR-FITC (BD,#555558), CD166-PE (BD,#560903), CD44-PE (BD,#555479), CD54-PEcy5 (BD,#555512), CD146-PE (BD,# 559263). At least 20,000 events were acquired on a BD FACSCalibur flow cytometer and data was analyzed using CellQuest software.
5
Characterizing Synovial MSC Phenotype
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Surface marker expression was analyzed by FACS Verse (BD) in synovial MSCs from four donors at passage 2. The cells before and 48 h after preservation were suspended in Hank’s balanced salt solution (HBSS) at a density of 5 × 105 cells/mL and stained for 30 min on ice with the antibodies CD44-PE-Cy7, CD73-V450, CD90-PE, CD105-APC, CD34-PE-Cy5, CD45-APC-H7, and CD31-FITC (all from BD), and Ghost Dye Violet 510 for dead cells (Tonbo Biosciences, CA, USA). These data were also analyzed using FlowJo software.
6
Comprehensive Mesenchymal Stem Cell Profiling
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After cell detachment using a 0.125% trypsin solution, cells were washed with PBS and resuspended in PBS containing 2% FBS. Cell concentration and viability were monitored using Trypan blue in a Neubauer hemocytometer. The following monoclonal antibodies were used as indicated by the manufacturers (BD Pharmingen® (BD, Franklin Lakes, New Jersey, USA)): CD90-PE (BD, #555596), CD73-FITC (BD, #561254), CD105-FITC (BD, #561443), CD45-FITC (BD, #347463), CD14-PE (BD, #555398), CD34-PEcy5 (BD, #561819), CD31-PE (BD, #555446), IgG-FITC (BD, #555786), HLA-DR-FITC (BD, #555558), CD166-PE (BD, #560903), CD44-PE (BD, #555479), CD54-PEcy5 (BD, #555512), CD146-PE (BD, #559263). Isotype controls were used for determining nonspecific binding and defining cut-off values. A minimum of 20,000 events were acquired on a BD FACS Calibur® flow cytometer and results were analyzed using CellQuest™ software.
7
Assessing Human Cell Engraftment
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Levels of human cell engraftment were assessed 18 weeks post-transplantation. Briefly, tibiae, femora, and pelvises were harvested from individual mice, and BM was flushed from each bone into 2 mL of Iscove's Modified Dulbecco's Medium (IMDM) using an insulin syringe with a 29G needle. Samples were then centrifuged at 1,200 rpm for 10 min. Red blood cells were lysed by 5- to 10-min incubation at room temperature with Ammonium-Chloride-Potassium (ACK) lysis buffer (Quality Biological), and the remaining cells were collected by centrifugation at 1,200 rpm for 10 min. Cells were stained for 30 min on ice with the antibody panel CD45 allophycocyanin (APC) (BD Pharmingen), CD20 phycoerythrin-cyanin 7 (PE-Cy7) (BD Biosciences), CD34 PE-Cy5 (BD Pharmingen), and CD13 PE (BD Pharmingen) and analyzed on a Fortessa flow cytometer (Becton Dickinson). Human engraftment was defined as more than 0.1% of CD45+ cells. Additionally, cells from the six mice in the rAAV6+RNP group were stained with CD45 APC (BD Pharmingen) and sorted by flow cytometry using BD FACSAria II or BD FACSAria Fusion instruments. Following sorting, cells were spun down at 3,000 rpm for 30 min, and genomic DNA was extracted as described previously. Genomic DNA was subjected to nested PCR using an in-out PCR approach to detect forward direction integration at the 5′ junction end (primer sequences are detailed in Table S4 ).
8
Characterization of MSCs from Different Sources
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To characterize MSCs derived from different sources (N-7 for each tissue of MSC isolation), the following conjugated antibodies were used: CD10 FITC, CD14 FITC, CD29 APC, CD44 FITC, CD45 PE-Cy7, CD73 PE, CD90 APC, CD105 AF488, CD146 PE, CD271 (NGFR) PE, (Exbio Antibodies, Vestec u Prahy, Czech Republic), VEGFR2, SSEA-4 PE (BioLegend, San Diego, CA, USA), CD133 PE, MSCA-1 APC (Miltenyi Biotec Inc., Bergisch Gladbach, Germany), CD34 PE-Cy5, HLA-ABC, HLA-DR (BD Bioscience, San Diego, CA, USA) and CD235a PE (Abcam, Cambridge, UK). As negative controls, mouse IgG1 and IgG2a conjugated with FITC, PE and PE-Cy5 (Dako Cytomation, Glostrup, Denmark, BD Bioscience, San Diego, CA, USA) were used. Briefly, cells from passage 3 were sampled in designated tubes of at least 250 000 cells in 100 µl, and incubated with the appropriate amount of antibodies according to the manufacturer’s recommendation. Cells were then washed with PBS, fixed in 4% paraformaldehyde overnight and at least 10 000 cells were recorded on BD FACSAriaTM Cell Sorter, BD LSR II (BD Bioscience, San Diego, CA, USA) or Apogee A-50 Micro flow cytometer (Apogee Flow Systems, Hertfordshire, UK). The obtained data were then analysed using FlowLogic™ Software (Inivai™ Technologies, Mentone, Australia).
9
Multiparametric Flow Cytometry Analysis of Human Cells
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Samples collected from 3D induction cultures or from the transplantation mice were isolated at time points indicated and washed in PBS supplemented with 2 % FBS. Cell clumps were removed by filtering the samples through 40-mm cell strainers, and the single-cell suspensions were incubated with mouse anti-human antigens. The following antibodies were used in the study: PE-Tra-1-85 (BD), PE-Cy5-CD34 (BD), PE-Cy7-CD34 (BioLegend), ECD-CD45 (BECKMAN COULTER), PE-CD45 (BioLegend), APC-CD45 (BioLegend), PE-Cy7-CD3 (BD), PE-Cy5-CD3 (BD), PE-Cy7-CD19 (BD), PE-Cy5-CD19 (BD), PE-Cy5-CD15 (BD), PE-Cy7-CD15 (BD), PE-CD235a (BD), PE-Cy7-CD71 (BD), FITC-CD144, FITC-CD117, FITC-CD43, PE-CD31, PE-CD38, and FITC-CD309. The incubation with the antibodies was carried out at room temperature for 30 min. The appropriate isotype IgGs (BD or BioLegend) served as controls. Cells were analyzed using a FlowCytometer FC500MCL, CXP Software, and FlowJo 7.6 Software. To ensure specificity, mouse samples, including the bone marrow, spleen, thymus, and peripheral blood samples, were stained with the same set of human antibodies as described above and the final data were reported with the background subtraction (data not shown here).
10
Quantification of Hematopoietic Progenitor Cells
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Freshly isolated blood-derived mononuclear cells and sputum-extracted cells were immediately incubated with fluorescence-labeled antibodies to define cell subpopulations. Antibodies (BD Biosciences) used for flow cytometry were FITC-CD45, PE-Cy5-CD34, and PE-CD125. Cells were analyzed by FACSVerse analytical flow cytometry (BD Company, San Diego, CA, USA). The percentage of HPCs (FSCmediumSSClowCD45dullCD34+) and EoPs (FSCmediumSSClowCD45dullCD34+CD125+) were determined using FlowJo software (BD Biosciences). The absolute numbers of HPCs or EoPs were calculated by multiplying percentage of HPCs or EoPs within R2 (singlets with medium FSC and low SSC) in flow cytometry with the absolute number of lymphocytes in blood routine test or sputum cell counts. The gating strategy is shown in Supplementary Figure S1
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