OVA323–339 peptide was synthesized by the Emory University Microchemical Core Facility. Ovalbumin was from Worthington Biochemical Corporation and hen egg lysozyme was from Sigma-Aldrich. Some reagents were purchased: mouse IFN-γ (PeproTech), and lisinopril (Sigma). The PE-conjugated anti-CD69, PE-conjugated anti-CD80 (16–10A1), APC-conjugated anti-CD3ε, APC-conjugated anti-CD86 (GL-1) and the ELISA kits for IL-2, IFN-γ and IL-17A were all purchased from eBioscience. Alexa Fluor 488-conjugated Ovalbumin was from ThermoFisher. AF6–120.1 (anti-I-Ab),1G11 (anti-EEA1) and eBioH4A3 (anti-LAMP-1) were from eBioscience. A rabbit antiserum that recognizes mouse ACE was previously described (13 (link)). The FITC-conjugated mouse TCR Vβ Screening Panel was from BD Pharmingen. Flow cytometry stained samples were analyzed on CyAn ADP (Beckman Coulter) and data were analyzed with FlowJo software.
Apc conjugated anti cd3ε
Manufactured by Thermo Fisher Scientific
Sourced in United States
The APC-conjugated anti-CD3ε is a monoclonal antibody that binds to the CD3 epsilon chain, a component of the T cell receptor complex. This antibody is conjugated with allophycocyanin (APC), a fluorescent dye, allowing for the detection and identification of T cells in flow cytometry applications.
Automatically generated - may contain errors
Lab products found in correlation
Percp conjugated anti cd45, by BioLegend (3 mentions)
Lisinopril, by Merck Group (2 mentions)
Hen egg lysozyme, by Merck Group (2 mentions)
Mouse ifn γ, by Thermo Fisher Scientific (2 mentions)
Elisa kits for il 2, by Thermo Fisher Scientific (2 mentions)
Pe conjugated anti cd69, by Thermo Fisher Scientific (2 mentions)
Pe conjugated anti cd80 16 10a1, by Thermo Fisher Scientific (2 mentions)
Apc conjugated anti cd86 gl 1, by Thermo Fisher Scientific (2 mentions)
Alexa fluor 488 conjugated ovalbumin, by Thermo Fisher Scientific (2 mentions)
Ebioh4a3 anti lamp 1, by Thermo Fisher Scientific (2 mentions)
Fitc conjugated mouse tcr vβ screening panel, by BD (2 mentions)
Cyan adp, by Beckman Coulter (2 mentions)
Ifn γ, by Thermo Fisher Scientific (2 mentions)
Ovalbumin (ova), by Worthington (2 mentions)
Rpmi 1640 medium, by Sartorius (2 mentions)
Fitc conjugated anti cd4, by Thermo Fisher Scientific (2 mentions)
Pacific blue conjugated anti cd8, by BioLegend (2 mentions)
Facscanto 2 flow cytometer, by BD (2 mentions)
Nylon cell strainer, by BD (2 mentions)
Fitc conjugated anti ly6g, by BD (2 mentions)
7 protocols using apc conjugated anti cd3ε
1
Ovalbumin Peptide Immunization Protocol
2
Ovalbumin Peptide Immunization Protocol
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OVA323–339 peptide was synthesized by the Emory University Microchemical Core Facility. Ovalbumin was from Worthington Biochemical Corporation and hen egg lysozyme was from Sigma-Aldrich. Some reagents were purchased: mouse IFN-γ (PeproTech), and lisinopril (Sigma). The PE-conjugated anti-CD69, PE-conjugated anti-CD80 (16–10A1), APC-conjugated anti-CD3ε, APC-conjugated anti-CD86 (GL-1) and the ELISA kits for IL-2, IFN-γ and IL-17A were all purchased from eBioscience. Alexa Fluor 488-conjugated Ovalbumin was from ThermoFisher. AF6–120.1 (anti-I-Ab),1G11 (anti-EEA1) and eBioH4A3 (anti-LAMP-1) were from eBioscience. A rabbit antiserum that recognizes mouse ACE was previously described (13 (link)). The FITC-conjugated mouse TCR Vβ Screening Panel was from BD Pharmingen. Flow cytometry stained samples were analyzed on CyAn ADP (Beckman Coulter) and data were analyzed with FlowJo software.
3
Lymphocyte Profiling by Flow Cytometry
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Blood, spleens, and PPs were used to analyze lymphocyte populations by flow cytometry. Cell suspensions from PPs and spleens were obtained by passing the tissues through a nylon cell strainer with a 40 μm pore size (BD Biosciences, San Jose, CA, USA) in RPMI1640 medium (Biological Industries, Kibbutz Beit Haemek, Israel). After lysing erythrocytes and centrifugation at 300 ×g for 10 min, pelleted cells were suspended in staining buffer. Whole-blood samples and cell suspensions from spleens and PPs were split into 100 μL aliquots and incubated with PerCP-conjugated anti-CD45 (Clone 30-F11, Biolegend, San Diego, CA, USA), APC-conjugated anti-CD3ε (Clone 145-2C11, eBioscience, San Diego, CA, USA), PE-conjugated anti-CD19 (Clone 6D5, Biolegend), FITC-conjugated anti-CD4 (Clone GK1.5, eBioscience), and Pacific blue-conjugated anti-CD8 (Clone 53-6.7, Biolegend) antibodies (Abs). Stained cells were analyzed with a FACS Canto II flow cytometer (BD Biosciences). CD45-positive cells were gated and considered to be leukocytes. Lymphocyte populations were determined as the percentages of T cells (CD3ε+CD19−) and B cells (CD3ε−CD19+) among leukocytes. Subpopulations of Th cells and cytotoxic T cells are presented as the percentage of CD4+CD8− and CD4−CD8+ cells among CD3ε-expressing cells.
4
Multiparameter Flow Cytometry of Leukocytes
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Flow cytometry was used to analyze the distribution of peripheral blood leukocytes and T cell subpopulation in whole blood. Each blood sample was divided into 100 μL in each aliquot, and these were then incubated with the different antibodies as described below. The antibodies used to detect leukocytes were as follows: PerCP-conjugated anti-CD45 (Biolegend, San Diego, CA, USA) for leukocytes, PE-conjugated anti-F4/80 (eBioscience, San Diego, CA, USA) for monocytes/macrophages and FITC-conjugated anti-Ly6G (BD Biosciences, San Jose, CA, USA) for neutrophils. For analysis of the T cell subpopulation, blood was incubated with PerCP-conjugated anti-CD45 (Biolegend), APC-conjugated anti-CD3ε (eBioscience), FITC-conjugated anti-CD4 (eBioscience) and Pacific blue-conjugated anti-CD8 (Biolegend) antibodies. The concentrations of antibodies were compliant with those recommended by the manufacturer. After 30 min of incubation at 4 °C in the dark, the lysed red blood cells were first suspended in staining buffer and then analyzed with a FACS Canto II flow cytometer (BD Biosciences). CD45-positive cells were gated for analysis, with the results presented as a percentage of specific CD-marker-expressing cells in blood leukocytes.
5
Immunophenotyping of Lymphocytes in Murine Mesenteric Lymph Nodes
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Cell suspensions from MLNs were obtained by passing the tissues through a nylon cell strainer with a 40 μm pore size (BD Biosciences) in RPMI1640 medium (Biological Industries, Kibbutz Beit Haemek, Israel). After centrifugation at 300 ×g for 10 min, pelleted MLN cells were suspended in 1 mL of staining buffer. One hundred microliters of cell suspension was incubated with APC-conjugated anti-CD3ε (eBioscience) and Pacific blue-conjugated anti-CD19 (Biolegend) for 30 min at 4°C in the dark. Stained cells were washed and resuspended in staining buffer to measure the lymphocyte population by flow cytometry. Percentages of T and B lymphocytes were determined by CD3ε- and CD19-expressing cells in MLN cells. Representative flow cytometry plots are shown in Figure 2(b) .
6
Multiparametric Flow Cytometry Analysis of Peripheral Blood Leukocytes
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A five-color flow cytometric analysis was performed to determine the distribution of peripheral blood leukocytes. Antibodies against mouse leukocyte surface antigens were added to 100 μL aliquots of whole blood. The antibodies used to detect different subsets of leukocytes were as follows: PerCP-conjugated anti-CD45 (Biolegend, San Diego, CA, USA) for leukocytes, PE-conjugated anti-F4/80 (eBioscience, San Diego, CA, USA) for monocytes/macrophages, FITC-conjugated anti-Ly6G (BD Biosciences, San Jose, CA, USA) for neutrophils, APC-conjugated anti-CD3ε (eBioscience) for T cells, and Pacific blue-conjugated anti-CD19 (Biolegend) for B cells. Antibodies were used at the concentration recommended by manufacturer. After a 30 min incubation at 4°C in the dark, red blood cells were lysed, and cells were suspended in staining buffer and then analyzed with a FACS Canto II flow cytometer (BD Biosciences). CD45-positive cells were gated, and results are presented as a percentage of specific CD-marker-expressing cells in blood leukocytes. Representative flow cytometry plots are shown in Figure 2(a) .
7
Flow Cytometry Analysis of Immune Cells
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LPMCs suspended in staining buffer (PBS containing 1 % FBS, 5 mM EDTA and 0.02 % NaN3) were incubated with anti-CD16/32 monoclonal antibodies (mAb) for 30 min at 4 °C to block nonspecific binding. Then, cells were stained for 30 min at 4 °C with a combination of the following fluorescent mAbs: FITC-conjugated anti-Ly6G or anti-DX5 (BD Biosciences), PE-conjugated anti-CD11c (BD Biosciences), PE-Cy7-conjugated anti-CD11b (BD Biosciences), Alexa Fluor 647-conjugated anti-Ly6C (BioLegend), APC-conjugated anti-CD3ε (eBioscience) or anti-CD19 (eBioscience). Dead cells were gated out by staining with 7-aminoactinomycin D (7-AAD) (Life Technologies, Carlsbad, CA). Stained cells were acquired using FACSCalibur (BD Biosciences) or FACSAriaIII (BD Biosciences), and data were analyzed with FlowJo software (TreeStar Inc., Ashland, OR).
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