Peripheral blood mononuclear cells (PBMCs) were isolated from 12 mL of peripheral venous blood samples collected in K3EDTA tubes (BD vacutainer) by centrifugation using a Ficoll-Paque gradient and were frozen at −80°C. Prior to flow cytometry analyses, cells were thawed, and PBMCs subtypes were distinguished using the following antibodies: anti-CD45-Pacific orange (Invitrogen), anti-CD3-V450 (BD Biosciences), anti-CD4-PE-texas red (Invitrogen), anti-CD8-APC-H7 (BD Biosciences), anti-CD19-PE-Cy7 (BD Biosciences), anti-CD14-PerCP-Cy5 (BD Biosciences), anti-CD16-FITC (BD Biosciences), anti-TLR2-PE (eBiosciences), and anti-TLR4-APC (eBiosciences) (all made in mouse). Data acquisition was performed using a BD LSR II flow cytometer, and the results were analyzed with BD eDiva Software (version 6.1.2, BD Biosciences) and FCS Express 4 Flow Cytometry (De Novo Software).
Anti cd45 pacific orange
Manufactured by Thermo Fisher Scientific
Anti-CD45-Pacific Orange is a monoclonal antibody conjugate that binds to the CD45 surface antigen. It is designed for use in flow cytometry applications to identify and analyze cells expressing the CD45 protein.
Automatically generated - may contain errors
Lab products found in correlation
Anti cd11b, by BioLegend (2 mentions)
Anti cd11c pe cy7, by BioLegend (2 mentions)
Apc efluor780 anti cd11b, by Thermo Fisher Scientific (2 mentions)
Alexafluor647 anti cd301, by BioLegend (2 mentions)
Live dead fix blue, by Thermo Fisher Scientific (2 mentions)
Anti f4 80 pe, by BioLegend (2 mentions)
Lung dissociation kit, by Miltenyi Biotec (2 mentions)
Gentlemacs dissociator, by Miltenyi Biotec (2 mentions)
Anti mouse cd16 32 fc block, by BD (2 mentions)
Fcs express 4 flow cytometry, by De Novo Software (1 mentions)
Anti cd19 pe cy7, by BD (1 mentions)
Anti cd4 pe texas red, by Thermo Fisher Scientific (1 mentions)
K3 edta tubes, by BD (1 mentions)
Anti tlr2 pe, by Thermo Fisher Scientific (1 mentions)
Anti cd3 v450, by BD (1 mentions)
Anti tlr4 apc, by Thermo Fisher Scientific (1 mentions)
Lsr 2 flow cytometer, by BD (1 mentions)
Anti cd16 fitc, by BD (1 mentions)
Anti cd8 apc h7, by BD (1 mentions)
Anti cd14, by Thermo Fisher Scientific (1 mentions)
6 protocols using anti cd45 pacific orange
1
Characterization of Peripheral Blood Immune Cells
2
Multiparameter Flow Cytometry of ILCs and T Cells
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Harvested and processed PBMCs were block and stained as follows. For the ILC panel, cells were stained with lineage markers (anti-CD3, anti-CD4, anti-CD8, anti-CD16, anti-CD14, anti-CD19 [eBioscience], anti-CD56, anti-FcεR1, anti-CD11b and anti-CD11c [Biolegend, San Diego, CA]), all labeled with Pacific Blue, anti-CD45-Pacific Orange (Invitrogen), anti-cKit-PerCP-eF710 (eBioscience) and anti-IL-7Rα-APC-Cy7 (eBioscience). Cells were then permeabilized and stained with anti-human IL-13-FITC (eBioscience) and data were collected on a BD FACS Canto II (BD Biosciences).
For the T cell panel, PBMCs were stained with anti-CD3-V500, anti-CD4-Qdot 605 and anti-CD8-PE-Texas Red. Cells were then fixed, permeabilized and stained with anti-IL-4-FITC, anti-IL-2-PerCP, anti-IL-10-Pacific Blue, anti-IL-22-APC, anti-TNF-α, anti-IL-17A-APC-Cy7, anti-IL-5-PE and anti-IFN-γ-PE-Cy7 for 30 minutes. Data were collected on a BD LSRFortessa (BD Biosciences). All flow data were analyzed using FlowJo version 9.4.10.
For the T cell panel, PBMCs were stained with anti-CD3-V500, anti-CD4-Qdot 605 and anti-CD8-PE-Texas Red. Cells were then fixed, permeabilized and stained with anti-IL-4-FITC, anti-IL-2-PerCP, anti-IL-10-Pacific Blue, anti-IL-22-APC, anti-TNF-α, anti-IL-17A-APC-Cy7, anti-IL-5-PE and anti-IFN-γ-PE-Cy7 for 30 minutes. Data were collected on a BD LSRFortessa (BD Biosciences). All flow data were analyzed using FlowJo version 9.4.10.
3
Identification and Characterization of Mast Cells
4
Isolation and Characterization of Lung Immune Cells
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Whole-lung single-cell suspensions were prepared as described previously (45 (link)). Briefly, lungs were minced and digested in buffer containing 2.2 mg/ml collagenase type IV (Worthington), 100 μg/ml DNase I (Zymo Research), and 5% fetal bovine serum (FBS) at 37°C rotating for 45 min. Digested samples were then passed through an 18-gauge needle, resuspended in red blood cell lysis buffer, diluted with PBS, passed through a 100-μm filter, and counted. For flow cytometry analysis, cells were stained with a viability dye (eFluor 780; eBioscience), anti-CD45 (Pacific Orange; Invitrogen), anti-CD11b (PECy5 [BioLegend] for cellularity, PerCPCy5.5 [BioLegend] for FLARE), anti-CD11c (phycoerythrin [PE], BioLegend), anti-Ly6G (fluorescein isothiocyanate [FITC], BioLegend), anti-CD64 (BV421; BioLegend), and anti-SiglecF (BV421; BD Bioscience). Samples were analyzed on a MACSQuant VYB flow cytometer (cellularity) or Beckman Coulter Cytoflex S (FLARE). Macrophages were identified as CD45+/Ly6G−/CD11b+/CD64+, neutrophils were identified as CD45+/SiglecF−/Ly6G+/CD11b+, and eosinophils were identified as CD45+/SiglecF+/CD11clow. Analysis was performed with FlowJo version 9.9.6.
5
Characterization of Lung Macrophages
6
Characterization of Lung Macrophages
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Mouse lungs were dissociated into single-cell suspensions using enzymatic digestion with the Lung Dissociation Kit (Miltenyi Biotec 130-095-927) and a GentleMACS Dissociator (Miltenyi Biotec) following the manufacturer’s protocol. One million cells per sample were incubated in Live/Dead Fix Blue (ThermoFisher L23105) for 20 minutes at 4°C and then washed and incubated with Fc Block (anti-mouse CD16/32) (BD Pharminogen 553142) for 10 minutes at room temperature and appropriate antibodies were added for 30 minutes at 4°C (Pacific Orange anti-CD45 (Invitrogen MCD4530), PE anti-F4/80 (BioLegend 123109), APC-eFluor780 anti-CD11b (Invitrogen 47-0112-82), PE-Cy7 anti CD11c (BioLegend 117317), and AlexaFluor647 anti-CD301 (BioLegend 145603)). Cells were then washed twice, fixed with 1% paraformaldehyde, and analyzed with the BD LSRII flow cytometer and FlowJo v10.7 software. For characterization of M1- and M2-polarized macrophage populations, CD45+F4/80highCD11bhigh populations were distinguished based on surface expression of CD11c (M1 marker) or CD301 (M2 marker)19 (link). Resident and recruited macrophage populations were defined as F4/80+CD11b−CD11c+ and F4/80+CD11b+CD11c− populations, respectively, as described previously20 (link).
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