The phenotype of rDFSCs and rDPCs was identified by flow cytometric analysis. The MSC phenotyping cocktail comprised both positive (CD29-FITC, CD44/CD90-PE, BD Bioscience, USA) and negative (CD34-PE, CD45-FITC, BD Bioscience, USA) fluorochrome-conjugated monoclonal antibodies. IgG1-FITC and IgG1-PE (BD Bioscience, USA) were used as isotype controls. Third-passage rDFSCs and rDPCs were suspended to 5 × 105 cells/mL in PBS solution, stained with different antibodies for 30 min at 4 °C, washed with PBS, resuspended in FACS buffer, and analyzed using a MOFlo™ high-performance cell sorter (Beckman Coulter, USA).
Igg1 pe
Manufactured by BD
Sourced in United States, Germany, United Kingdom
IgG1-PE is a fluorescently-conjugated immunoglobulin G1 (IgG1) antibody used in flow cytometry and other immunoassays. It serves as a detection reagent for the quantification and analysis of target antigens.
Automatically generated - may contain errors
Lab products found in correlation
Igg1 fitc, by BD (26 mentions)
Igg1 apc, by BD (11 mentions)
Facscalibur, by BD (9 mentions)
Cd34 pe, by BD (8 mentions)
Cd45 fitc, by BD (6 mentions)
Cellquest software, by BD (5 mentions)
Igg2a fitc, by BD (5 mentions)
Cd4 fitc, by BD (4 mentions)
Cd44 pe, by BD (4 mentions)
Cd73 pe, by BD (4 mentions)
Bovine serum albumin (bsa), by Merck Group (4 mentions)
Facsdiva software, by BD (4 mentions)
Igg2a apc, by BD (3 mentions)
Hla dr fitc, by BD (3 mentions)
B220 fitc, by BD (3 mentions)
Cd27 pe cy7, by BD (3 mentions)
Sodium azide, by Merck Group (3 mentions)
Igg2a pe, by BD (3 mentions)
Accuri c6, by BD (3 mentions)
Accuri c6 flow cytometer, by BD (3 mentions)
61 protocols using igg1 pe
1
Phenotyping rDFSCs and rDPCs by Flow Cytometry
2
Breast Cancer Stem Cell Isolation
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To detect the BCSCs subpopulations, the following antibodies were applied: anti-CD44-APC, anti-CD24-PE, IgG1-PE, IgG1-APC (BD). Human breast specimens were mechanically dissociated and incubated with 200 U/ml Liberase Blendzyme 4 (Roche, USA) for 2 h to obtain single cell suspensions. Then cell staining and flow cytometry were performed as described previously16 (link). Cells were sorted on a flow cytometer (FACSAriaII, BD, USA) and analyzed on another flow cytometer (C6, BD, USA) with BD FACS Diva software.
3
Flow Cytometric Analysis of T-Cell Subsets
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Fresh peripheral venous blood was collected from all patients 1 day before biopsy. Anticoagulant (100 μL) was added to each of the two test tubes containing either 20 μL CD4-FITC (BD, Franklin Lakes, NJ, USA) or 20 μL CD8-PE (BD) and mixed evenly. Additionally, a test tube with blood from Control mice and IgG1-FITC (BD) or IgG1-PE (BD) monoclonal antibodies (20 μL) was also mixed. All test tubes were incubated at room temperature after mixing for 20 min; then, 2 mL of red cell lysate (BD Biosciences, San Jose, CA, USA) was added. After incubation at room temperature for 10 min, samples were centrifuged at 12,000 rpm for 5 min, the supernatant was discarded, and 2 mL of phosphate buffer pH 7.4 (0.05% PBS) was added. The samples were mixed well and washed 1–2 times. Cells were resuspended in 500 μL of PBS, and immediately tested by flow cytometry. We gated for small lymphocytes (R1 gate) based on the forward scatter and side scatter of the cells. Mouse IgG1-FITC and IgG1-PE monoclonal antibodies were used as isotype-negative controls.
The data were acquired using a FACScan flow cytometer (BD) and analyzed with CellQuest software (BD).
The data were acquired using a FACScan flow cytometer (BD) and analyzed with CellQuest software (BD).
4
Comprehensive Immune Cell Profiling
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Extracellular surface marker staining was performed with IgG1-FITC, IgG2a-FITC, IgG1-PE, αCD25-FITC, αCD40-PE, αCD69-PE, αCD70-PE, αCD80-FITC, αCD83-PE, αCD86-FITC, and PD-L1-PE (all from BD Biosciences, Heidelberg, Germany); IgG3-PE (eBioscience, Frankfurt, Germany); and αCCR7-FITC (R&D Systems, Minneapolis, MN, USA) for 30 min at 4 °C in PBS supplemented with 1% FCS and 0.02% sodium azide (Merck, Darmstadt, Germany). The cells were analyzed using a FACScan cytofluorometer equipped with CellQuest software (BD, Heidelberg, Germany). Analysis was performed with the FCS Express software (De Novo Software, Glendale, CA, USA). Specific MFIs were calculated by subtracting the background MFI obtained with the isotype controls.
5
Phenotypic characterization of hDPSCs
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Flow cytometric analysis was conducted to identify the phenotype of hDPSCs. The MSC phenotyping cocktail is comprised of both positive (CD29-FITC, CD44-PE, and CD90-PE-CY5, BD Bioscience, USA) and negative (CD34-PE and CD45-PE-CY5, BD Bioscience, USA) fluorochrome-conjugated monoclonal antibodies. IgG1-FITC, IgG1-PE-CY5 and IgG1-PE (BD Bioscience, USA) were used as isotype controls. hDPSCs were suspended to 4 × 105 cells/mL, incubated with different antibodies for 30 min at 4 °C, resuspended in FACS buffer, and analyzed using a MOFloTM high-performance cell sorter (Beckman Coulter, USA).
6
IFNγ Modulates ASC Surface Markers
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ASC were grown in normal or 3 ng/mL IFNγ conditions for 48 h. ASC were then trypsinized and counted for a final concentration of 50,000 ASC per 100 μL autoMACS Running Buffer (Miltenyi). For antibody staining we used CD46 (564253, BD), CD55 (MCA1614PE, Serotec), and CD59 (BRA-10G, Novus Biologicals) Abs and their respective isotypes as controls (IgG2a-APC, IgG1-PE, and IgG2b-PE from BD). After 20 min ice incubation ASC were washed with autoMACS Running Buffer (Miltenyi) and centrifuged 500 × g for 4 min. Finally, ASC were resuspended in 100 μL autoMACS Running Buffer (Miltenyi) transferred to cytometry tubes acquired in a LSR Fortessa flow cytometer (BD) and analyzed with BD FacsDiva™ (BD).
7
Flow Cytometric Analysis of T-Cell Subsets
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Lymphocytes were labeled for 30 min with 5 µL of the respective monoclonal antibodies: anti-CD3 PerCP, anti-CD4 FITC, and anti-CD8 P.E. (B.D. Biosciences, San Jose, CA, USA). An isotype control (IgG1-FITC or IgG1-PE (B.D. Biosciences, San Jose, CA, USA) was used in all analyses, and cells underwent flow cytometry (FACSCalibur, BD Bioscience, San Jose, CA, USA).
8
Immunophenotyping of Cell Populations
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Fluorescence activated cell sorting (FACS) was carried out as described previously in our paper [20 (link)]. The following antibodies were used to mark the cell surface epitopes: CD90-phycoerythrin (PE), CD44-PE, CD73-PE, CD166-PE and CD34-PE, CD45-fluoroisothyocyanate (FITC), and HLA-DR-FITC (all from BD Pharmingen). All analyses were standardized against negative control cells incubated with isotype specific IgG1-PE and IgG1-FITC (BD Pharmingen). At least 10,000 events were acquired on Guava Technologies flow cytometer, and the results were analyzed using Cytosoft, Version 5.2 (Guava Technologies).
9
Identification of CD44+ and CD133+ Cells
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Two million cells were harvested from 85% confluent flasks and resuspended in PBS with 0.1% BSA. The cells were washed and incubated at 4°C for 30 minutes with anti-CD44-allophycocyanin (APC) (1:20 dilution, clone G44-26, BD Biosciences) and anti-CD133-phycoerythrin (PE) (1:20 dilution, clone AC133, MiltenyiBiotec) antibodies, or mouse-specifc IgG2b ĸ-APC (1:100dilution, BD Biosciences) and IgG1-PE (1:20 dilution, Miltnyi Biotec) antibodies. The cells were then washed and resuspended in PBS with 0.1% BSA and 2 μg/mL propidium iodide (PI), and a C6 FACS (BD Biosciences) was used for all analyses. The cells were first gated on the basis of side-scatter and forward-scatter, followed by the exclusion of nonviable (PI-positive) cells. The CD44+ and CD133+ gates were created on the basis of cellular staining with the isotype control antibodies (IgG2bĸ-APC and IgG1-PE, respectively).
10
PBMNC and QQMNC Characterization by FACS
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To characterise PBMNCs and QQMNCs (n = 6 per group), fluorescence-activated cell sorting (FACS) analysis was performed using a BD LSRFortessa Cell Analyzer (BD Biosciences) and CellQuest software (BD Biosciences) after staining with mouse anti-human monoclonal antibodies against the following surface markers: Cluster of Differentiation (CD)34-Brilliant Violet (BV) 421 (clone 581; BD Biosciences); Vascular Endothelial Growth Factor Receptor(VEGFR)2-Phycoerythrin(PE) (clone 7D4-6; BioLegend, San Diego, California); Cluster of Differentiation (CD206)-fluorescein(FITC) (clone 15-2; BioLegend); and C-C chemokine receptor type 2 (CCR2)- Brilliant Violet (BV)605 (clone K036C2; BioLegend). Dead cells were excluded on the basis of 7-Amino-Actinomycin D (7-AAD) staining (BD Biosciences). Cells were stained with monoclonal antibodies for 20 minutes at 4°C following Fc receptors (FcR) blocking, washed twice using Hank’s buffered salt solution containing 2% fetal bovine serum (FBS), and then analysed. Relevant isotype controls (Immunoglobulin G(IgG)1-BV421 isotype control (BD Biosciences), IgG1-PE (BD Biosciences), IgG1-FITC (BD Biosciences), and IgG2a-BV605 (BioLegend)) were also used. In all samples, 20 000 events were acquired.
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