Cd8 apc cy7

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CD8-APC-Cy7 is a fluorescence-labeled antibody that binds to the CD8 cell surface protein, which is expressed on a subset of T cells. It can be used for the identification and enumeration of CD8+ T cells in flow cytometry applications.

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CD8-APC (44 citations) APC-Cy7 (32 citations) Anti-CD8-APC-Cy7 (4 citations)

14 protocols using cd8 apc cy7

Multiparametric Flow Cytometry Profiling of PBMCs

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PBMCs were counted and allowed to rest for 2 h in FACS buffer (PBS containing 10% FCS, 5 mM EDTA and 2 mM azide). For each staining 1×10⁶ PBMCs were used. The staining includes the following markers: CD3-PECy5.5 (Beckman Coulter, USA), CD4-PECy7 (Biolegend, USA), CD8-APCCy7 (BD Biosciences, USA), CD45RA-eFluor605 (eBiosciences, USA), CD57-Pacific Blue (Biolegend), CD28⁻PETexasRed (Beckman Coulter), CD27⁻APC (Biolegend). All staining performed included a Live/Dead marker (Invitrogen, USA) to exclude false positive staining. The CD4/CD8 ratio was measured in whole blood to match with previous studies.^{16 (link)} Briefly, 100 μl whole blood were stained with CD3-PECy5.5, CD4-PECy7 and CD8-APCCy7 for 20 min at 4 degrees followed by incubation with lysis buffer (eBiosciences) to eliminate red blood cells. Flow Cytometry data were analyzed using FlowJo (Treestar, USA), FACSDiva (BD Biosciences) and Kaluza (Beckman Coulter) (Supplementary Figure S1).

Ng T.P., Camous X., Nyunt M.S., Vasudev A., Tan C.T., Feng L., Fulop T., Yap K.B, & Larbi A. (2015). Markers of T-cell senescence and physical frailty: insights from Singapore Longitudinal Ageing Studies. NPJ Aging and Mechanisms of Disease, 1, 15005-.

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Multiparametric Flow Cytometry of Immune Cells

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Flow cytometric assay was done as previously described.^{52 (link),53 (link)} Briefly, bone marrow cells isolated by flushing the tibias and femurs of the euthanized mice, resuspended in a single-cell suspension and undiluted whole blood cells were used for flow cytometric analysis. Following the removal of red blood cells, the remaining cells were stained with a cocktail of antibodies (CD11b (Mac-1)-PE, Gr-1-PECy7, CD68 APC, CD4-eFluoro450 (PB), B220APC, and CD8-APCCy7 from eBiosciences) to identify the population of myeloid, B cells, and T cells. Flow cytometry analysis was performed on a Becton Dickinson 5-laser LSRII instrument.

West J., Chen X., Yan L., Gladson S., Loyd J., Rizwan H, & Talati M. (2020). Adverse effects of BMPR2 suppression in macrophages in animal models of pulmonary hypertension. Pulmonary Circulation, 10(1), 2045894019856483.

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Comprehensive Immune Cell Profiling Protocol

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The cells were stained with monoclonal antibodies to T-cell phenotype CD3 APC, CD4 PerCP Cy 5.5, CD8 APC Cy7, CD27 FITC, CD45RA PE (eBioscience, CA, USA). The cells were also stained with monoclonal antibodies to MDSC phenotype CD3 APC, CD19 APC, CD56 APC, HLA-DR APC e-fluor 780, CD33 PerCP Cy5.5, CD11b PE, CD14 PE Cy7, CD15 FITC (eBioscience, CA, USA). After 30 min of incubation with monoclonal antibodies, in the dark and at 4°C, the cells were washed with PBS and centrifuged. Living cells (based on forward and side scatter) were acquired in the FACS Canto II using the DIVA software (Becton Dickinson, USA). Further analyses of FACS data were performed using the 9.3 FLOWJO software (Tree Star, USA).
T lymphocytes were characterized as described previously (36 (link)).

Naïve: CD3⁺CD4⁺CD45RA⁺CD27⁺ or CD3⁺CD8⁺CD45RA⁺CD27⁺ (Naïve).

Central memory: CD3⁺CD4⁺CD45RA⁻CD27⁺ or CD3⁺CD8⁺CD45RA⁻CD27⁺ (CM).

Effector memory: CD3⁺CD4⁺CD45RA⁻CD27⁻ or CD3⁺CD8⁺CD45RA⁻CD27⁻ (EM).

Effector memory re-expressing CD45RA: CD3⁺CD4⁺CD45RA⁺CD27⁻ or CD3⁺CD8⁺CD45RA⁺CD27⁻ (EMRA).

Myeloid-derived suppressor cells were characterized as:

CD3⁻CD19⁻CD56⁻HLA⁻DR^−/lowCD33⁺CD11b⁺CD15⁺ granulocytic or

CD3⁻CD19⁻CD56⁻HLA⁻DR^−/lowCD33⁺CD11b⁺CD14⁺ monocytic.

Alves A.S., Ishimura M.E., Duarte Y.A, & Bueno V. (2018). Parameters of the Immune System and Vitamin D Levels in Old Individuals. Frontiers in Immunology, 9, 1122.

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Multiparameter Flow Cytometry Analysis

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Viable cells were detected by LIVE/DEAD (L/D) Fixable Aqua stain (Invitrogen) and the following antibodies were used for surface and intracellular staining: CD3ɛ Pacific Blue, CD4 FITC, CD8 APC-Cy7, IFN-γ APC, LAMP1 PE, TNF-α PE (eBioscience), PE-labeled H-2Kb/SIINFEKL pentamer (ProImmune). The following steps were performed on ice. Cells were washed with PBS, stained for 15 min with Aqua L/D stain, resuspended in PBS with 2% FBS for surface staining for 15 min and fixed for 15 min in PBS + 2% PFA. IFN-γ intracellular staining was performed in PBS + 2% FBS supplemented with 0.5% saponin. Flow cytometry was performed using a CyAn ADP Analyzer (Beckman Coulter). Detection of HBsAg and IFN-γ were performed using an HBsAg ELISA kit (AMS biotechnology) and a Ready-Set-Go ELISA Kit (eBioscience), respectively, according to the manufacturers protocol.

Julier Z., de Titta A., Grimm A.J., Simeoni E., Swartz M.A, & Hubbell J.A. (2016). Fibronectin EDA and CpG synergize to enhance antigen-specific Th1 and cytotoxic responses. Vaccine, 34(21), 2453-2459.

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Flow Cytometry Immunophenotyping of Immune Cells

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Flow cytometry measurements were performed using a CyAn ADP Analyzer (Beckman Coulter) and data were analyzed using version 9.8.2 of FlowJo software. Viable cells were detected by LIVE/DEAD (L/D) Fixable Aqua stain (Invitrogen) and the following antibodies were used for surface and intracellular staining: CD3e Pacific Blue, CD4 FITC, CD8 APC-Cy7, CD45.1 Pe-Cy7, CD25 PE, PD-1 PE, CTLA4 APC, IFNγ APC, FOXP3 PerCP-Cy5.5 (eBioscience). For staining of blood and splenocytes, cells were exposed for 5 min at RT to 0.155 NH₄Cl to lyse erythrocytes. The following staining steps were performed on ice. Cells were washed with PBS, stained for 15 min with Aqua L/D stain, resuspended in PBS + 2% FBS for surface staining for 15 min and finally fixed for 15 min in PBS + 2% PFA. IFNγ intracellular staining was performed in PBS + 2% FBS supplemented with 0.5% Saponin. Foxp3 Transcription Factor Fixation/Permeabilization Concentrate and Diluent kit (ebioscience) was used for FOXP3 staining.

Grimm A.J., Kontos S., Diaceri G., Quaglia-Thermes X, & Hubbell J.A. (2015). Memory of tolerance and induction of regulatory T cells by erythrocyte-targeted antigens. Scientific Reports, 5, 15907.

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Comprehensive Immune Cell Profiling

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Peripheral blood was collected by facial vein bleed into EDTA Microtainer tubes (BD Biosciences, NJ) and CBC performed on a Heska Hematrue (Heska, CO). Flow cytometry was acquired on a FACSVerse (BD Biosciences, NJ) and analyzed using Flowjo (Flowjo, OR). Cell sorting was performed on an Astrios (Beckman Coulter). Antibodies were used from (1) BioXCell: Fc block (2.4G2), and (2) eBioscience: B220-APC-Cy7 (RA3–6B2), CD11c-APC (N418), CD138-PE-Cy7 (DL-101), CD19-APC (1D3), CD19-PE-Cy7 (1D3), CD21-FITC (4E3), CD23-PE (B3B4), CD25-APC (PC61.5), CD3-FITC (145–2C11), CD4-PE-Cy7 (RM4–5), CD43-APC (R2/60), CD44-PE (IM7), CD45.1-PE (A20), CD45.2-APC (104), CD5-APC (53–7.3), CD62L-APC (MEL-14), CD8-APC-Cy7 (53–6.7), CD8-FITC (53–6.7), Foxp3-PE (FJK-16s), IFNγ-APC (XMG1.2), IgD-FITC (11–26c), IgM-PE (eB121–15F9), IL-17-APC (eBio1787), NK1.1-PB (PK136). Cell viability assessed using fixable Live/Dead Aqua dye (Invitrogen). Flow cytometry for LC3 was performed per manufacturer’s instructions (FlowCellect, Millipore).

Khor B., Conway K.L., Omar A.S., Biton M., Haber A.L., Rogel N., Baxt L.A., Begun J., Kuballa P., Gagnon J.D., Lassen K.G., Regev A, & Xavier R.J. (2019). Distinct tissue-specific roles for the disease-associated autophagy genes ATG16L2 and ATG16L1. Journal of immunology (Baltimore, Md. : 1950), 203(7), 1820-1829.

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Single Cell Isolation and Immunophenotyping

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In order to obtain single cell suspensions, spleen and mesenteric lymph node (MLN) tissues were passed through 70μm filters in a PBS solution supplemented with 3% fetal bovine serum. Bone marrow (BM) was flushed from the long bones of mice with 22 ½ gauge syringes using 3% FBS in PBS, and then subsequently passed through 70μm filters. Red blood cells were then removed from the single cell suspensions by lysis with Ammonium-Chloride-Potassium buffer.
An equal number of cells for each sample were first blocked with FcγR for 20 minutes, followed by one wash with PBS and incubation with the primary antibody (1:100–1:500) for 30 minutes at room temperature in the dark. When secondary antibodies were necessary, samples were incubated with the conjugated antibody for 30 minutes after primary antibody incubation. Samples were then washed once with PBS, transferred to FACS tubes, and passed through either BD LSR II flow cytometer or Fortessa FACS machines for analysis. Data were analyzed using FlowJo software v8.8.2 (Tree Star, Ashland, OR). Antibodies were purchased from eBioscience (San Diego, CA): c-kit APC #17-1171-82, Stem cell antigen-1 (Sca) PE-Cy7 #25-5981-82, Myeloid differentiation antigen (Gr1) APC-Cy7 #47-5931-82, Macrophage-1 antigen (Mac1) APC-Cy7 #47-0112-82, B220 APC-Cy7 #47-0452-82, CD4 APC-Cy7 #47-0042-82, CD8 APC-Cy7 #47-0081-82.

Danielson L.S., Reavie L., Coussens M., Davalos-Vega V., Castillo-Martin M., Guijarro M., Coffre M., Cordon-Cardo C., Aifantis I., Ibrahim S., Liu C., Koralov S, & Hernando E. (2014). Limited miR-17-92 overexpression drives hematologic malignancies. Leukemia research, 39(3), 335-341.

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Flow Cytometric Analysis of Immune Responses

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A total of 1 × 10⁶ cells in BALs from immunized and challenged mice were stained with extracellular (anti-mouse CD3e-FITC, CD4-APC, CD8-APC-Cy-7, CD69-PECy-5) (eBioscience, CA, United States) markers using established procedures (32 (link)). For intracellular cytokine staining, 2 × 10⁶ splenocytes were cultured for 4 days with medium only, peptide pools (1 μg/ml of each lipopeptide) or PPD (1 μg/ml). On day 5, brefeldin A (1.5 μg/ml, 1 X; eBioscience) was added, and cultured for 5 h at 37°C and subsequently stained for extracellular markers CD3-PE Cy7, CD4-PE Cy5, CD8-APC-Cy7, and intracellular cytokines IFN-γ-PE and IL-10-FITC using our previously reported procedures. For intracellular GrB staining, 2 x 10⁶ splenocytes obtained from mice were stained for extracellular markers CD3-PE Cy7, CD49b-Alexaflour 700, CD8-APC-Cy7, and intracellular GrB-Alexaflour 647, without ex vivo stimulation, using our previously reported procedures (31 (link)). Samples were run on LSR Fortessa SORP flow cytometer and analyzed using FACS-DIVA software (Becton Dickinson, Mountain View, CA, United States). Respective isotype-matched control antibodies were used to gate non-specific staining in each experiment. Gates were set to exclude 95% of isotype control antibody stained cells in all extracellular and intracellular staining experiments.

Gupta N., Garg S., Vedi S., Kunimoto D.Y., Kumar R, & Agrawal B. (2018). Future Path Toward TB Vaccine Development: Boosting BCG or Re-educating by a New Subunit Vaccine. Frontiers in Immunology, 9, 2371.

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Phenotypic Characterization of Autoimmune T Cells

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Surface-antigen staining was performed according to standard protocols using the following antibodies: CD3-PerCpCy5.5 (BioLegend), CD4-PB (BioLegend), CD8-APC-Cy7 (eBioscience), CD25-PE (BD Bioscience), CD44-PeCy7 (BioLegend), CD62L-APC (eBioscience), anti-TCR-Vβ antibodies (BD Bioscience). For intracellular cytokine staining, 1 × 10⁶ lymphocytes were re-stimulated with 50 μg/ml myelin oligodendrocyte-derived glycoprotein (MOG)_33–55 peptide in the presence of Golgi Plug (BD Bioscience) for 6 h. The staining was performed with Cytofix/Cytoperm reagents (BD Bioscience) according to the manufacturer’s protocol. The following antibodies were used: IL-17-APC (BD Biosciences), GM-CSF-APC (BD Biosciences), IFNγ-APC (BD Biosciences). Intracellular transcription factor staining was performed using a FoxP3 staining buffer set (eBioscience) and a FoxP3-APC antibody (eBioscience) according to the manufacturer’s protocol. Flow cytometry was performed on a FACSCanto (BD Bioscience) with FACS Diva software (BD Bioscience) and analyzed with FlowJo 9.6 (Treestar).

Meister M., Papatriantafyllou M., Nordström V., Kumar V., Ludwig J., Lui K.O., Boyd A.S., Popovic Z.V., Fleming T.H., Moldenhauer G., Nawroth P.P., Gröne H.J., Waldmann H., Oelert T, & Arnold B. (2015). Dickkopf-3, a Tissue-Derived Modulator of Local T-Cell Responses. Frontiers in Immunology, 6, 78.

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Multiparameter Flow Cytometry Analysis

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CD3e-APC or CD3e-PE (clone 145-2C11), CD4-APC/Cy7 or CD4-PerCp (clone GK1.5), CD8α-APC/Cy7 or CD8α-PacBlue (clone 53–6.7), CD11c-APC or CD11c-PE (clone N418), CD11b-PerCP (clone M1/70), CD19-PerCP/Cy5.5 (clone HIB19), CCR4-APC or CCR4-PE/Cy7 (clone 2G12), CD25-PE (clone PC61), IFN-γ-Pacific Blue (clone XMG1.2), and Foxp3-Pacific Blue (clone MF-14) were all from BioLegend. T regs were stained with a Foxp3 Staining Buffer Set (eBioscience) according to the manufacturer’s instructions. For intracellular IFN-γ staining, peripheral blood or splenocytes were incubated with red blood cell lysis buffer (BD Pharm Lyse; BD Bioscience) for 3 min. Lymphocytes were then stimulated with 100 nM OVA_257–264 peptide or OVA_323–339 peptide (both InvivoGen) for 1 h at 37°C before 1 µg/ml brefeldin A (Sigma-Aldrich) was added. After 3 h, cells were surface stained with CD8-APC/Cy7 and CD19-PerCP, then fixed and permeabilized using the Foxp3 Staining Buffer Set (eBioscience) and incubated with an anti–IFN-γ-Pacific Blue antibody. Pentamer staining was performed with H-2Kb OVA_257–264 R-PE pentamers (ProImmune) according to the manufacturer’s instructions. Events were measured on a FACS Canto II flow cytometer (BD Biosciences) and analyzed with FlowJo software (TreeStar).

Rapp M., Wintergerst M.W., Kunz W.G., Vetter V.K., Knott M.M., Lisowski D., Haubner S., Moder S., Thaler R., Eiber S., Meyer B., Röhrle N., Piseddu I., Grassmann S., Layritz P., Kühnemuth B., Stutte S., Bourquin C., von Andrian U.H., Endres S, & Anz D. (2019). CCL22 controls immunity by promoting regulatory T cell communication with dendritic cells in lymph nodes. The Journal of Experimental Medicine, 216(5), 1170-1181.

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