7 color immunophenotyping kit

Manufactured by Miltenyi Biotec

Sourced in Germany

The 7-color Immunophenotyping kit is a laboratory equipment designed for the analysis of cell surface markers using flow cytometry. It allows the simultaneous detection of up to 7 different cellular markers in a single sample, providing a comprehensive immunophenotypic profile of cells.

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4 protocols using 7 color immunophenotyping kit

PBMC Cell Proportions Analysis

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Given that PBMCs are a mixture of white blood cells, cell proportions need to be considered when analyzing DNA methylation data [17 (link)]. We used an aliquot of 0.5 × 10⁶ of the thawed PBMCs to measure cell proportions by staining with the 7-color Immunophenotyping kit (Miltenyi Biotech, Gladbach, Germany) and analyzing them by flow cytometry (Fortessa, Becton Dickinson, NJ, USA). Detailed information on antibodies is described in Table S1. The antibody staining was done for 30 min at 4 °C in the dark. After staining, cells were washed with 2 mL FACS buffer (350× g, 8 min, 4 °C) re-suspended in 200 µL FACS buffer and kept on ice. For every sample, at least 5000 events were detected After gating based on CD45⁺ cells, cell proportions for T cells, B cells, monocytes, and neutrophils were expressed as a percentage of the total sample. All statistical analyses for cell count and individual variables were performed using the R-software version 3.0 (https://www.r-project.org/). The Kruskal–Wallis test was used for comparison of cell counts between the three different lifestyle groups and t-test (unpaired) for comparison between sensitized and non-sensitized individuals. A p value < 0.05 was considered as statistically significant.

Acevedo N., Scala G., Merid S.K., Frumento P., Bruhn S., Andersson A., Ogris C., Bottai M., Pershagen G., Koppelman G.H., Melén E., Sonnhammer E., Alm J., Söderhäll C., Kere J., Greco D, & Scheynius A. (2021). DNA Methylation Levels in Mononuclear Leukocytes from the Mother and Her Child Are Associated with IgE Sensitization to Allergens in Early Life. International Journal of Molecular Sciences, 22(2), 801.

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Comprehensive Immune Cell Profiling

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The immunophenotyping of the donor PBMC was performed by the direct immunofluorescence method using the “7-Color Immunophenotyping Kit” (Miltenyi Biotec, Bergisch Gladbach, Germany). Sample preparation was carried out according to the manufacturer’s instructions. Cells stained with immunofluorescence-labelled antibodies were analyzed using a MACSQuant Analyzer 10 flow cytometer (Miltenyi Biotec, Bergisch Gladbach, Germany), to determine the following cell subpopulations: CD45⁺ (leukocytes), CD45⁺ CD3⁺ (T cells), CD45⁺ CD3⁺ CD4⁺ (T-helper cells), CD45⁺ CD3⁺ CD8⁺ (CTL), CD45⁺ CD19⁺ (B cells), CD45⁺ CD14⁺ (monocytes), SSC^lowCD45⁺ CD14^-CD16⁺ CD56⁺ CD3⁻ (NK-cells), SSC^highCD45⁺ CD14⁻CD16⁻ (eosinophils), SSC^highCD45⁺ CD14⁻CD16⁺ (neutrophils).
The results of immunophenotyping of PBMC from healthy donors [43 (link)] as well as the reference values used in clinical practice were used as reference values in the current study.

Konopleva M.V., Borisova V.N., Sokolova M.V., Semenenko T.A, & Suslov A.P. (2022). Recombinant HBsAg of the Wild-Type and the G145R Escape Mutant, included in the New Multivalent Vaccine against Hepatitis B Virus, Dramatically Differ in their Effects on Leukocytes from Healthy Donors In Vitro. Vaccines, 10(2), 235.

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Quantification of Human Hematopoietic Cells

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A Miltenyi MacsQuant 10 instrument was used for flow cytometric analysis of bone marrow hematopoietic cell populations. To assess for human hematopoietic stem and progenitor cell populations the following antibodies purchased from Biolegend were used: FITC antimouse CD45 (103108), APC antihuman CD45 (368512), Pacific Blue antihuman Lineage Cocktail (348805; CD3, CD14, CD16, CD19, CD20, CD56), PE antihuman CD34 (343506), Brilliant Violet 510 antihuman CD38 (356612), and 7-AAD Viability Staining Solution (420404). Human lineage analysis was performed using the 7-color immunophenotyping kit (130–098-456; Miltenyi Biotec).

Doan P.L., Frei A.C., Piryani S.O., Szalewski N., Fan E, & Himburg H.A. (2023). Cord Blood–Derived Endothelial Progenitor Cells Promote In Vivo Regeneration of Human Hematopoietic Bone Marrow. International journal of radiation oncology, biology, physics, 116(5), 1163-1174.

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Immunophenotyping of Whole Blood in GCA

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Venous blood was drawn from GCA patients into heparin-containing tubes. Whole blood immunophenotyping was performed using 7-Color Immunophenotyping kit with the following antibodies (Miltenyi Biotec, catalog #130-098-456): CD14-FITC (clone Tük4), CD56-PE (clone REA196), CD16-PE (clone REA423), CD4-PerCP (clone VIT4), CD19-PE-Vio® 770 (clone LT19), CD3-APC (clone BW264/56), CD8-APC-Vio 770 (clone BW135/80), CD45-VioBlue® (clone 5B1). Briefly, 100 μl of whole blood was incubated with 10 μl immunophenotyping reagent for 10 min in the dark, at 4°C. After incubation, whole blood was lysed using Red Blood Lysing Solution (Miltenyi Biotec, catalog #130-098-456). Neutrophil phenotyping was performed in 50 μl of whole blood, incubated for 30 min at 4°C in the dark, with the following antibodies (eBioscience): CD16-PE (clone eBioCB16; catalog #50-112-4738), CD62L-PE-Cy5 (clone DREG56; catalog #50-140-71) and CD11b-APC (clone ICRF44; catalog #17-0118-42). After incubation, samples were lysed, using Whole Blood Lysing Reagent Kit (Beckman Coulter; catalog #6602764). All samples were analyzed using flow cytometer MACSQuant Analyzer 10 (Miltenyi Biotec). Analysis of flow cytometry data was performed using MACSQuantify (Analysis Software version 2.8, Miltenyi Biotec) and FlowLogic (Flow Cytometry Analysis Package, version 7.00.0a, Invasion Software Technologies Pvt Ltd).

Kuret T., Frank-Bertoncelj M., Lakota K., Žigon P., Thallinger G.G., Kopitar A.N., Čučnik S., Tomšič M., Hočevar A, & Sodin-Šemrl S. (2022). From Active to Non-active Giant Cell Arteritis: Longitudinal Monitoring of Patients on Glucocorticoid Therapy in Combination With Leflunomide. Frontiers in Medicine, 8, 827095.

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