Cd8 pc7

Manufactured by Beckman Coulter

Sourced in United States

The CD8-PC7 is a flow cytometry reagent used for the detection and enumeration of CD8+ T cells. It contains an antibody conjugated to the PC7 fluorochrome, which binds to the CD8 cell surface antigen, allowing for the identification of CD8-positive cells in a sample.

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6 protocols using cd8 pc7

Multiparametric Flow Cytometry Panels for Immune Activation and Exhaustion

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Two panels were designed; Immune Activation Panel and Immune Exhaustion Panel. The Immune Activation Panel consisted of the following fluorochrome-coupled monoclonal antibodies: CD3-FITC; CD4-ECD; CD8-PC-7; CD45-KO; CD38-PE and; HLA-DR-APC (Beckman Coulter, MI). The Immune Exhaustion Panel was composed of CD3-FITC; CD4-ECD; CD8-PC7; CD45-KO; CTLA-4-PE and; PD-1-APC. Flow cytometry data acquisition was performed using a Navios flow cytometer (Beckman Coulter). Optimal volatges for acquisition were determined, compensation and fluorescence minus one analyses were performed before testing of study samples. The gating strategy is shown in Additional file 1: Figure S1. Data analysis was performed using Beckman Coulter Kaluza software.

Maponga T.G., Andersson M.I., van Rensburg C.J., Arends J.E., Taljaard J., Preiser W, & Glashoff R.H. (2018). HBV and HIV viral load but not microbial translocation or immune activation are associated with liver fibrosis among patients in South Africa. BMC Infectious Diseases, 18, 214.

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Flow cytometric analysis of PBMC responses

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The available number of PBMC allowed further analysis of eight samples by flow cytometry. In detail, 0.5 × 10⁶ cells were transferred to a 96-well round-bottom plate in 100 µL culture medium with fetal calf serum in the presence of the corresponding peptide pool or SEB or culture medium alone. Following 1-h incubation at 37 °C in a humidified 5% CO2, brefeldin A (Sigma-Aldrich) at a final concentration of 10 ug/mL was added. After overnight incubation, cells were washed in phosphate-buffered saline (PBS) ethylenediaminetetraacetic acid (EDTA) 2mM and incubated with the Live/Dead Fixable Far Red Dead Cell Stain Kit (ThermoFisher Scientific, Waltham, MA, USA) for 30 min at 4 °C. Cells were then washed with PBS, fixed and permeabilized with BD Cytofix/Cytoperm (BDBiosciences, San Jose, CA, USA) according to the manufacturer’s instructions for intracellular staining with the following monoclonal antibodies: IFNγ-FITC, CD4-ECD, CD8-PC7, CD3-Pe-Cy5 (all from BeckmanCoulter, Brea, CA, USA). Finally, the cells were resuspended in 4% paraformaldehyde and analyzed with a Navios flow cytometer (Beckman Coulter, Brea, CA, USA), obtaining the percentages of IFN-γ secreting lymphocyte cell responses and allowing phenotypical discrimination of individual cytokine-producing cells

Cassaniti I., Ferrari A., Comolli G., Sarasini A., Gregorini M., Rampino T., Lilleri D, & Baldanti F. (2021). Characterization of Varicella-Zoster (VZV) Specific T Cell Response in Healthy Subjects and Transplanted Patients by Using Enzyme Linked Immunospot (ELISpot) Assays. Vaccines, 9(8), 875.

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Comprehensive Flow Cytometry Analysis

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Analysis of cluster differentiation markers was achieved by flow cytometry with a Gallios analytical flow cytometer (Beckman Coulter, Irving, TX, USA). Aliquots (100 µL) of peripheral blood were mixed with the following fluorochrome-conjugated antibodies: Anti-human CD3-APC, CD4-APCeF750, CD8-PC7, CD14-PC5.5, CD16-PB, CD45-KrO, CD54-FITC, and CD56-PE (Beckman Coulter, Irving, TX, USA), and lysed with the Beckman Coulter Versalyse solution following the manufacturer’s recommendations. To obtain the cell counts for the different populations 100 µL of Flow-Count Fluorospheres (Beckman Coulter, Irving, TX, USA) were added.

Gómez de Cedrón M., Laparra J.M., Loria-Kohen V., Molina S., Moreno-Rubio J., Montoya J.J., Torres C., Casado E., Reglero G, & Ramírez de Molina A. (2019). Tolerability and Safety of a Nutritional Supplement with Potential as Adjuvant in Colorectal Cancer Therapy: A Randomized Trial in Healthy Volunteers. Nutrients, 11(9), 2001.

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Comprehensive T-cell Immunophenotyping by Flow Cytometry

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CD4 and CD8 T-lymphocyte phenotypes were determined on fresh whole blood, using combinations of the following antibodies: CD28-FITC, CD4-RD1, CD45RA-ECD, CD45RO-ECD, CD62L-PC5, CD27-PC5, CD4-PC7, and CD8-PC7 (Beckman Coulter, Villepinte, France), as described in [18 (link)]. CD4_N levels were defined as the percentage of CD4 T lymphocytes that were CD45RA⁺CD62L⁺, CD8_N levels as the percentage of CD8 T lymphocytes that were CD45RO^-CD28⁺CD27⁺. For 57 patients, frozen PBMCs were available for the quantification of CD4_RTE, with Live-Dead-Aqua (Life Technologies, Saint-Aubin, France) labeling of dead cells, and the following antibodies: CD3-ECD and CD31-FITC (Beckman Coulter), CD4-APC-efluor780 (e-bioscience, Paris, France), CD45RA-V450 and CD197-PC7 (BD Biosciences, Rungis, France). CD4_RTE levels were defined as the percentage of naive CD45RA⁺CCR7⁺CD4⁺ T lymphocytes positive for CD31.

Le Chenadec J., Scott-Algara D., Blanche S., Didier C., Montange T., Viard J.P., Dollfus C., Avettand-Fenoel V., Rouzioux C., Warszawski J, & Buseyne F. (2015). Gag-Specific CD4 and CD8 T-Cell Proliferation in Adolescents and Young Adults with Perinatally Acquired HIV-1 Infection Is Associated with Ethnicity — The ANRS-EP38-IMMIP Study. PLoS ONE, 10(12), e0144706.

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T cell Proliferation Analysis by Flow Cytometry

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A six-colour flow cytometry panel was designed to specifically analyse the proliferation of T cells. Therefore, cells were pipetted through a 40 µm cell strainer (Cat# 43-10040-70, pluriSelect, Leipzig, Germany) and stained with CD45 FITC (Cat# A07782, Beckman Coulter, Krefeld, Germany), 7-AAD (Cat# A07704, Beckman Coulter, Krefeld, Germany), CD4 PC7 (Cat# 737660, Beckman Coulter, Krefeld, Germany), CD8 PC7 (Cat# 737661, Beckman Coulter, Krefeld, Germany) and CD5 APC (Cat# 345783, BD Biosciences, Heidelberg, Germany). For PBMCs freshly isolated on day 0, cells were incubated with 1 mL of 1× NH₄Cl-based erythrocyte lysing solution (contained in Cat# IM3630d, Beckman Coulter) for 10 min at room temperature. Analysis was performed using the FACSLyric Flow Cytometer and the FACSuite software 1.4 (BD Biosciences, Heidelberg, Germany).
In order to determine the extent of T cell proliferation, a gating strategy was established for CD45⁺/7AAD⁻/CD5⁺/CD4⁺ or CD8⁺ singlet cells. Those cells are further referred to as T cells. Then, the proportion of T cells that had divided at least once (fraction diluted, Dil as described by Roederer et al. [31 (link)]) was determined and the number of daughter generations arising from the initial population was counted. Mean values for each triplicate were calculated to determine the inhibition by MSCs using the following formula:

Piede N., Bremm M., Farken A., Pfeffermann L.M., Cappel C., Bonig H., Fingerhut T., Puth L., Vogelsang K., Peinelt A., Marschalek R., Müller M., Bader P., Kuçi Z., Kuçi S, & Huenecke S. (2023). Validation of an ICH Q2 Compliant Flow Cytometry-Based Assay for the Assessment of the Inhibitory Potential of Mesenchymal Stromal Cells on T Cell Proliferation. Cells, 12(6), 850.

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Multiparametric PBMC Immunophenotyping

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To determine percentages of T helper lymphocytes, T cytotoxic lymphocytes, natural killer cells, B cells and monocytes, 50,000 PBMCs were stained for 15 min at room temperature in tube A containing staining buffer (PBS, containing 0.2% BSA, 0.1% sodiumazide, pH 7.8) with CD45-Pacific Orange (Invitrogen, Carlsbad, CA, USA) 1:80, CD3-PercP-Cy5.5 (BD Biosciences, San Jose, CA, USA) 1:16, CD4-Pacific Blue (BD Biosciences) 1:200, CD8-PC7 (Beckman Coulter, Brea, CA, USA) 1:80, CD19-APC (BD Biosciences) 1:80, CD14-APC-H7 (BD Biosciences) 1:16, CD56-PE (Cytognos, Salamanca, Spain) 1:40 and CD15-FITC (BD Biosciences) 1:400.

Counotte J., Drexhage H.A., Wijkhuijs J.M., Pot-Kolder R., Bergink V., Hoek H.W, & Veling W. (2018). Th17/T regulator cell balance and NK cell numbers in relation to psychosis liability and social stress reactivity. Brain, behavior, and immunity, 69.

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