One million cells were suspended in 500 µl FACS staining buffer (0.2% BSA fraction V and 0.09% Sodium azide in PBS) containing 20 µg/ml antibodies. After incubation for 60 min at 4°C, the cells were washed with PBS and resuspended in 1 ml FACS staining buffer for flow cytometric analysis. Fluorescein isothiocyanate (FITC), phycoerythrin (PE), peridinin chlorophyll protein-Cy5.5 (PerCP-Cy5.5), or Alexa Fluor 647-coupled antibodies against CD11b, CD44, CD45, CD73, CD90, and CD105 (Becton Dickinson) were used. For isotype controls, FITC-, PE-, PerCP-Cy5.5-, or Alexa Fluor 647-coupled nonspecific rat immunoglobulin G (IgG; Becton Dickinson) was substituted for the primary antibody. Cell fluorescence was evaluated by flow cytometry using a FACSVerse instrument (Becton Dickinson). The data were analyzed using FACSuite software (Becton Dickinson).
Percp cy5
Manufactured by BD
Sourced in United States, Germany, United Kingdom
PerCP-Cy5.5 is a fluorescent dye that can be used in flow cytometry applications. It has excitation and emission wavelengths that allow it to be detected using commonly available flow cytometry instrumentation.
Automatically generated - may contain errors
Lab products found in correlation
Fluorescein isothiocyanate (fitc), by BD (15 mentions)
Apc cy7, by BD (12 mentions)
Lsrfortessa, by BD (7 mentions)
Facscanto 2, by BD (6 mentions)
Facscanto, by BD (5 mentions)
Facscanto 2 flow cytometer, by BD (4 mentions)
Facsuite software, by BD (4 mentions)
Facsaria, by BD (4 mentions)
Facsdiva software, by BD (4 mentions)
Apc cy7, by BioLegend (3 mentions)
Facsaria 3, by BD (3 mentions)
Fc block, by BD (3 mentions)
Golgistop, by BD (3 mentions)
Cytofix cytoperm reagent, by BD (3 mentions)
γδ tcr, by BD (2 mentions)
Annexin 5, by BD (2 mentions)
Live dead aqua, by Thermo Fisher Scientific (2 mentions)
Fcs express, by De Novo Software (2 mentions)
Cd20 apc cy7, by BD (2 mentions)
Cd45 phycoerythrin pe cy7, by BD (2 mentions)
120 protocols using percp cy5
1
Immunophenotyping of Cell Suspensions
2
Profiling PD-1 and LAG-3 expression on T cells
3
Multiparametric Phenotypic Profiling of PBMCs
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Cryopreserved PBMCs (>90% viability) were thawed in supplemented RPMI-1640 medium (10% human serum, penicillin/ streptomycin (Invitrogen), 2 mmol/l l -glutamine (Invitrogen, Carlsbad, California, USA)), before being stained on ice for 30 minutes as previously described [6 (link)]. Cells prepared for analysis of total CD4+ and CD8+ populations were stained with fluorochromatic antibodies provided by BD Bioscience using 2.5 μl of CD3 (PE), CD4 (PE-Cy7), and CD8 (APC-Cy7) antibodies. For analysis of CD4+ subpopulations, 2.5 μl of CD3 (APC; BD Bioscience), 2.5 μl of CD4 (PerCP-Cy5.5; BD Bioscience), 1 μl of CD45RA (APC-Cy7; BioLegend), 3 μl of CCR7 (PE-Cy7; BD Bioscience), and 1.5 μl of CD27 (PE; BD Bioscience) antibodies was used. Analysis of CD8+ T cell subpopulations used a similar staining strategy to the CD4+ T cell subpopulation method, with the substitution of 2.5 μl of CD3 (PerCP-Cy5.5; BD Bioscience) in place of the APC version and 2.5 μl of CD8 (APC; BD Bioscience) in place of the CD4 to gate the total CD8+ T cell population. All stained cells prepared were washed and resuspended in 300 μl of 1× PBS prior to flow analysis or prior to subsequent staining with mitochondrial dyes.
4
Characterization of T-cell Cytokine Profiles
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Cells were stimulated with 50 ng/mL phorbol 12-myristate 13-acetate (PMA; Sigma) and 1 μM ionomycin (Sigma) with transport inhibitor GolgiStop (BD Biosciences, San Jose, CA, USA) in a cell incubator with 5% CO2, at 37 °C, for 4 h. Cells were transferred to tubes and washed with PBS. Cells were stained with anti-human CD4 antibodies conjugated with FITC (BD Biosciences, San Jose, CA, USA) for 30 min, at 4 °C. After fixation and permeabilization, cells were incubated with anti-human IFNγ antibodies conjugated with PerCP-Cy5.5, anti-human IL-4 antibodies conjugated with APC, and anti-human FoxP3 antibodies conjugated with PerCP-Cy5.5 (BD Biosciences, San Jose, CA, USA) for 30 min, at 4 °C. Isotype controls were used to correct nonspecific binding. Cells were analyzed with BD FACS Canto II flow cytometer (Becton, Dickinson and Company, Franklin Lakes, NJ, USA), and data were analyzed with FlowJo software (Becton, Dickinson and Company).
5
Quantification of Human Mesenchymal Stem Cells
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MSCs were detached from the plastic using TrypLETM Select 10× and resuspended at a concentration of 1 × 107 cells/mL in BD Pharmingen™ Stain Buffer. They were added to labelled tubes with the antibodies from the BD Stemflow™ Human MSC Analysis Kit: FITC Mouse Anti-Human CD90 (5 µL); PE Mouse Anti-Human CD44 (5 µL); PerCP-Cy™ 5.5 Mouse Anti-Human CD105 (5 µL); APC Mouse Anti-Human CD73 (5 µL); hMSC Positive Isotype Control Cocktail (20 µL); PE hMSC Negative Isotype Control Cocktail (20 µL); hMSC Positive Cocktail (20 µL) containing CD90 FITC, CD105 PerCP-Cy™ 5.5, CD73, and APC; and PE hMSC Negative Cocktail (20 µL) containing CD34 PE, CD11b PE, CD19 PE, CD45 PE, and HLA-DR PE. Volumes of 100 µL of the prepared cell suspension were added to the tubes outlined in step 2. The tubes were incubated in the dark for 30 min on ice. The cells were washed twice with BD Pharmingen™ Stain Buffer and resuspended at 300–500 µL in BD Pharmingen™ Stain Buffer and analyzed on a flow cytometer.
6
Characterization of Lung-Derived Extracellular Vesicles
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Cells, isolated from whole lung or MPs and EXs as described above, were stained with the following FITC- or PE- or PerCP-Cy5.5 labeling antibodies to CD3, CD4, CD8, NK1.1, γδTCR, CD69 and Annexin V (BD Pharmigen) and analyzed by Attune® Acoustic Focusing Cytometer (Thermo Scientific-Applied Biosystems, Foster City, CA, USA). Isotype control was used for all the samples. MPs and EXs were defined as Annexin V positive cells. To measure the size of MPs and EXs, we used calibration beads from 0.1 to 3 µm in diameter, as previously described. 48 (link), 49 (link)
7
Characterization of Lung-Derived Extracellular Vesicles
8
Detailed Multicolor Flow Cytometry Protocol
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Spleens, inguinal lymph nodes, blood and peritoneal fluids were isolated from WT T505A mice. For blood samples, red blood cell lysis was performed using Pharm Lyse lysis buffer (BD) according to manufacturer's instructions. Following the generation of single-cell suspensions, total cell counts were calculated in the presence of trypan blue (Sigma–Aldrich) using a haemocytometer, followed by incubation with anti-CD16/CD32 Fc-Blocking antibody (clone 2.4G2 (BD Pharmingen)). Distinction between live and apoptotic/necrotic cells was performed based on staining with LIVE/DEAD Aqua (Life Technologies). For cell surface marker detection cells were stained with a combination of FITC, PE, APC, PerCP-Cy5.5, APC-Cy7 conjugated monoclonal antibodies (BD Pharmigen). For detection of Foxp3, intracellular staining was performed using a Fix/Perm kit (eBiosciences). See below for additional antibody information. All samples were acquired using a three laser FACS Canto II (BD Bioscience) and the data were subsequently analysed using FlowJo (version 9 or X; Treestar, U.S.A.).
Antibodies used in flow cytometric analysis of lymphoid tissue cells
9
Mouse Spleen Lymphocyte Isolation and Activation
10
Multiparametric Flow Cytometry of Lymphocyte Populations
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