Unstimulated and IFNγ-stimulated AT-MSC were trypsinized and washed with FACS Flow (BD Biosciences, San Jose, CA). Cell suspensions were incubated with mouse-antihuman monoclonal antibodies against CD13-PE-Cy7; HLA-DR-PERCP; HLA-ABC-APC; CD31-FITC; CD73-PE; PD-L1-PE (all BD Biosciences); CD90-APC and CD105-FITC (R&D Systems, Abingdon, UK) at room temperature in the absence of light for 30 min. After two washes with FACS Flow, flow cytometric analysis was performed using FACSCANTO-II with FACSDIVA Software (BD Biosciences).
Mouse anti human monoclonal antibodies
Manufactured by BD
Sourced in United Kingdom, United States
Mouse anti-human monoclonal antibodies are laboratory reagents used to detect and quantify the presence of specific human proteins or antigens in biological samples. They are produced by hybridoma technology, where mouse B cells are fused with myeloma cells, resulting in a stable cell line that secretes antibodies targeting a particular human antigen.
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Lab products found in correlation
Cd13 pe cy7, by BD (1 mentions)
Hla abc apc, by BD (1 mentions)
Cd31 fitc, by BD (1 mentions)
Cd90 apc, by R&D Systems (1 mentions)
Cd105 fitc, by R&D Systems (1 mentions)
Hla dr percp, by BD (1 mentions)
Facscanto 2, by BD (1 mentions)
Facsdiva software, by BD (1 mentions)
Facsflow, by BD (1 mentions)
Cd73 pe, by BD (1 mentions)
Pd l1 pe, by BD (1 mentions)
Guava easycyte 8ht, by Merck Group (1 mentions)
Flow cytometry staining buffer, by R&D Systems (1 mentions)
Guava easycyte 8ht flow cytometer, by Merck Group (1 mentions)
Histopaque 1077, by Merck Group (1 mentions)
Phosphate buffered saline (pbs), by Merck Group (1 mentions)
Facscan flow cytometer, by BD (1 mentions)
Carboxyfluorescein succinimidyl ester (cfse), by Thermo Fisher Scientific (1 mentions)
Facsaria 3, by BD (1 mentions)
Cd25 pe, by BD (1 mentions)
12 protocols using mouse anti human monoclonal antibodies
1
Immunophenotypic Analysis of AT-MSCs
2
Phenotypic Characterization of hMSCs
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The immune‐phenotype of the hMSCs used was assessed by multi‐parameter flow cytometry using a previously developed protocol.29 Briefly, the cells were harvested from the substrate as described previously and counted, and the cell suspension was concentrated to achieve a concentration of 1 × 106 cells mL−1. 200 μL of cell suspension was then loaded on to a V‐bottom 96‐well plate and centrifuged at 220 × g for 5 min. The supernatant was aspirated, and the cell pellet was resuspended in cell staining buffer (R&D Systems, Abingdon, UK) and centrifuged again. The cells were stained with mouse anti‐human monoclonal antibodies (BD Biosciences, Swindon, UK) for 30 min in the dark at room temperature. The fluorescently labelled antibodies were selected based on a panel recommended by the International Society for Cell Therapy (ISCT);30 these were CD73 (PE‐Cy7), CD90 (APC), CD105 (PE), CD34 (PE‐Cy5) and HLA‐DR (FITC). The stained samples were analysed on a Guava EasyCyte 8HT flow cytometer (Merck Millipore, Watford, UK) equipped with 488 nm and 640 nm excitation lasers. A minimum of 10 000 gated events were recorded for each sample. Post‐acquisition analysis and compensation were performed using FlowJo software v8 (Treestar Inc., Meza, AZ, USA).
3
Quantifying Sputum Neutrophils by Flow Cytometry
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The concentration of neutrophils in sputum samples was determined by adding 100 μL diluted sputum to a TrueCount tube (BD 340334; Becton Dickinson). In addition, 5 μL of each of the following mouse anti-human monoclonal antibodies (BD Bioscience) were added to the samples: peridinin chlorophyll A protein-labelled CD14 PerCP (BD345786), FITC-labelled CD15 (BD5554019), and allophyocyanin (APC)-labelled CD45 (BD555485) (Bioscience, La Jolla, CA, USA). The samples were incubated for 30 min and fixated with 1 mL 1Xfluorescence activated cell sorter (FACS) lysis solution (BD349202) (Bioscience). Subsequently, the samples were stored overnight in the dark at 5°C before analysis by flow cytometry using a FACSCanto (Bioscience) equipped with a 15 mV argon-ion laser tuned at 488 nm, and a red diode laser emitting at 635 nm for excitation. Light-scatter and exponentially amplified fluorescence parameters from at least 10 000 events were recorded in list mode. Leukocytes were discriminated by gating on CD45 and the PMNs were discriminated by gating on CD14 and CD15. The instrument was calibrated using Calibrite beads (BD 349502, Bioscience) (S1 Fig ).
The neutrophils were counted by flowcytometry as previously described [16 (link)] within 14 days.
The neutrophils were counted by flowcytometry as previously described [16 (link)] within 14 days.
4
Immunophenotyping of Expanded Human Mesenchymal Stem Cells
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Immuno‐phenotype analysis was performed before and after hMSC expansion on the FC40/DMEM interface. hMSC immune‐phenotype was determined by multi‐parameter flow cytometry by using a previously developed protocol.3 Briefly, cells were harvested from the substrate as described previously, counted and the cell suspension concentrated to achieve 1 × 106cells mL−1. 200 μL of cell suspension was then loaded onto a V‐bottom 96 well plate and centrifuged at 220 g for 5 min. The supernatant was then aspirated, the cell pellet resuspended in flow cytometry staining buffer (R&D Systems, UK) and centrifuged again. The cells were stained with mouse anti‐human monoclonal antibodies (BD Biosciences, UK) for 30 min in the dark at room temperature. The fluorescently‐labelled antibodies were selected based on a panel recommended by the International Society for Cell Therapy (ISCT)22 and these were: CD73 (PE‐Cy7), CD90 (APC), CD105 (PE), CD34 (PE‐Cy5) and HLA‐DR (FITC). After incubation, the samples were washed twice with staining buffer as described before. The stained samples were then analysed on the Guava EasyCyte 8HT flow cytometer (Merck Millipore, UK) equipped with 488 nm and 640 nm excitation lasers. A minimum of 10 000 gated events were recorded for each sample. Post‐acquisition analysis and compensation were performed using the FlowJo software (Treestar Inc, USA).
5
Isolation and Analysis of PBMCs
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Fresh blood samples were centrifuged at 750 g for 10 min and the plasma fraction was removed and stored at −80 °C. The buffy coat was removed, diluted with 1% BSA in PBS (Sigma-Aldrich Chemical Co., St Louis, MO, USA), layered on a Histopaque 1077 (Sigma-Aldrich) polysaccharide-sodium diatrizoate density gradient, and spun at 600 g for 30 min. The cells at the gradient interface were removed, washed, and then counted using Trypan blue exclusion for use in flow cytometry. Flow cytometry studies were performed using the following mouse anti-human monoclonal antibodies (BD Pharmingen, Mississauga, ON, Canada): CD3-FITC, CD4-PE, CD8-PE, CD45RO-FITC, CD45RA-QR, CD62L-APC, CD14-APC, CD20-FITC, CD16-PE, CD25-PE, and CD56-QR. Approximately 106 cells were aliquoted into pretreated V-well plates, incubated for 20 min in 2% human albumin to block Fc receptors, and then incubated for 30 min with antibodies, both steps occurring in the dark at 4 °C. Cells were washed twice with FACS buffer (4% FBS in PBS), fixed in 1% paraformaldehyde, and stored at 4 °C until analysed on a FACScan flow cytometer (BD Biosciences, Sunnyvale, CA, USA). Gating was done on forward/side scatter plots to isolate the peripheral blood mononuclear cell (PBMC) fraction and 104 cells were counted per patient. Matched isotype controls were used for each fluorochrome to determine thresholds for positive expression.
6
Flow Cytometry Immunophenotyping of T Cells
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The following mouse anti-human monoclonal antibodies were purchased from BD Biosciences (San Diego, CA): anti-CD3 APC (HIT3a), anti-CD25 PE (M-A251), anti-CD4 PerCP (SK3), and anti-CD8 FITC (HIT8a). An IL-2 secretion assay detection kit was obtained from R&D Systems, Inc. (Quantikine ELISA, Minneapolis, MN), and carboxyfluorescein succinimidyl ester (CFSE) was purchased from Molecular Probes (Eugene, OR).
7
Immune Cell Characterization by FACS
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Each cryopreserved PBMC sample from the ZPHI study participants contained approximately 10 million cells. The samples were thawed, washed with PBS, and stained with the following fluorescently conjugated mouse anti-human monoclonal antibodies (Becton Dickinson): CD3–Pacific Blue (clone UCHT1), TCRαβ-FITC (clone T10B9.1A-31), CD4–Alexa Fluor 700 (clone RPA-T4), CD8-PE-CF594 (clone RPA-T8), CD14-APC-H7 (clone MΦP9), CD16-APC (clone 3G8), CD25-PE (clone M-A251), CD69-PE-Cy7 (clone FN50), and HLA-DR-PE-Cy7 (clone G46-6). Cells were subsequently separated by FACS (FACSAria III, Becton Dickinson) into the following cell populations: resting CD4+ T cells (CD3+, CD4+, CD25–, CD69–, and HLA-DR–), activated CD4+ T cells (CD3+, CD4+, and positive for at least 1 of these markers: CD25, CD69, or HLA-DR), CD4–/lo T cells (TCRαβ+, CD3+, and CD4–/lo), and monocytes (CD14+ and CD16+/–). 7-AAD (Becton Dickinson) staining was used to exclude dead cells. The proportion of sorted cells from PBMCs was similar to that previously observed (Supplemental Figure 4 and ref. 59 (link)).
8
Analysis of Circulating Lymphocyte Subsets
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The analysis of circulating lymphocyte subpopulations was performed on whole blood samples collected into heparinized tubes before the initiation of treatment. Heparinized blood (100 μL) was mixed with 20 μL of each of the following fluorescent mouse anti-human monoclonal antibodies according to the reagent instructions: CD3, CD4, CD8, CD19, CD25, CD44 and CD56 (Becton Dickinson, San Jose, CA). The mixtures were incubated for 30 minutes in the dark room, washed with phosphate buffered saline (PBS) (3 mL), and centrifuged at 1500 rpm for 5 minutes. The supernatant was discarded, and the pellet was resuspended with PBS (1 mL) for flow cytometric analysis. The percentage of fluorescent-positive cells was calculated with flow cytometry (Becton Dickinson). The experiments were repeated three times and the mean value was calculated for statistical analysis [10 (link)].
9
Monoclonal Antibody Microarray Printing
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Arrays were printed in a Microgrid printer with solid pins (Total array Systems, BioRobotics) on hydrogel coated slides (Full Moon Biosystems) using a panel of 231 monoclonal mouse anti-human antibodies (BD biosciences). The antibodies were printed at a concentration of 0.5 mg/ml in five spots, each using a single stamp and with 750 μm spacing. Following printing, the arrays were hydrated in a humidifier at 4°C for 48 hours, and then dried for 10 minutes at room temperature.
10
Isolation and Characterization of Early EPCs
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EPCs were cultured and characterized as detailed previously (Zhang et al., 2014 (link)). Briefly, peripheral blood mononuclear cells from hypertensive subjects were cultured on fibronectin-coated six-well plates in endothelial cell basal medium-2 (EBM-2; Clonetics, San Diego, CA, United States) supplemented with endothelial growth medium-SingleQuots (contents: ascorbic acid 0.5 mL; rhFGF-B 2.0ML; heparin 0.5 mL; GA-1000 0.5 mL; rhEGF 0.5 mL; hydrocortisone 0.2 mL; VEGF 0.5 mL; R3-IGF-1 0.5 mL). Early EPCs were defined as cells dually positive for DiI-acLDL (0.02 mg/mL; Invitrogen, Carlsbad, CA, United States) uptake and FITC-labeled BS-1 lectin (0.01 mg/mL; Sigma-Aldrich, St. Louis, MO, United States) binding as previously described (Xia et al., 2017 (link)). Endothelial marker proteins of cultured EPCs were also examined by flow cytometry analysis by using phycoerythrin–labeled monoclonal mouse anti-human antibodies recognizing CD31 (BD Pharmingen), von Willebrand factor (vWF) (BD Pharmingen), and kinase-insert domain receptor (KDR) (R&D System). Furthermore, expression of the monocytic lineage marker CD14 (BD Pharmingen) was analyzed as previously described. Based on the isolation and cultivation protocol, the adherent mononuclear cells were identified as early EPCs.
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