Cells were incubated for 5 minutes at room temperature with Fc block, and DAPI was added prior to analysis to assess viability. Flow cytometric analysis was performed on an LSR II cytometer (BD Biosciences). Data were acquired using CellQuest software (BD Biosciences) and analyzed using FlowJo software (Tree Star). For tumor infiltrating T cells, cells were gated on strict forward and side scatter parameters to ensure single cell analysis. Additionally, the DAPI- population was used for viable cells. Finally, cells were assessed for CD3 and CD4 or CD8 expression. The FACS antibodies used were: APC anti-mouse MHC Class I (H-2Kb) (Cat No. 17-5958-82) and PE-Cyanine 7 anti-Mouse CD45 (Cat No. 25-0451-82) were purchased from eBioscience, PE CD274 (B7-H1, PD-L1) anti-mouse mAb (clone MIH5) (Cat No. A14764) was from Life Technologies, Biotin anti-mouse CD95 (Cat No. 554256), anti-mouse CD16/CD32 (Mouse BD Fc Block™) (Cat No. 553142) PE anti-mouse CD3 (Cat No. 553063), FITC anti-mouse CD4 (Cat No. 553729) and PerCP-Cy5.5 anti-mouse CD8 (Cat No. 551162), were from BD Biosciences. Cell culture supernatants were tested for presence of IFNγ using a CBA assay (BD Biosciences).
Percp cy5.5 anti mouse cd8
Manufactured by BD
Sourced in United States
PerCP-Cy5.5 anti-mouse CD8 is a fluorescently-conjugated monoclonal antibody designed to detect the CD8 surface marker on mouse cells. It can be used in flow cytometry applications.
Automatically generated - may contain errors
Lab products found in correlation
Anti mouse cd16 cd32 mouse fc block, by BD (1 mentions)
Cba assay, by BD (1 mentions)
Anti mouse cd3 pe, by BD (1 mentions)
Fitc anti mouse cd4, by BD (1 mentions)
Cellquest software, by BD (1 mentions)
Lsrii cytometer, by BD (1 mentions)
Apc anti mouse cd45, by BD (1 mentions)
Anti mouse cd3 fitc, by BD (1 mentions)
5 and 6 chloromethyl 2 7 dichlorodihydrofluorescein diacetate cm h2dcfda, by Thermo Fisher Scientific (1 mentions)
Anti mouse cd4 pe, by BD (1 mentions)
Lsr 2, by BD (1 mentions)
Facsdiva, by BD (1 mentions)
2 protocols using percp cy5.5 anti mouse cd8
1
Multiparametric Flow Cytometry of Tumor-Infiltrating Immune Cells
2
Quantifying Immune Cell Phenotypes and ROS
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To analyze the ROS positive cells and T cell phenotypes (CD4 or CD8 positive), the isolated PBLs were incubated with antibodies labeled with fluorochromes in a fluorescence-activated cell sorting (FACS) buffer (1% FBS and 0.05% sodium-azide contained PBS) for 30 min on ice, washed twice with the FACS buffer, measured using a BD LSR II (BD Bioscience, San Jose, CA, USA), and analyzed using BD FACS Diva (BD Biosciences) or FlowJo software (Three Star Inc., Ashland, OR, USA). The antibodies used for the immune cell phenotyping were as follows: FITC-anti-mouse-CD3, PE-anti-mouse-CD4, PerCP-Cy5.5-anti-mouse-CD8, and APC-anti-mouse CD45 (BD Pharmingen, SanDiego, CA, USA). The intracellular ROS production was estimated by fluorescence using 5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate (CM-H2DCFDA; Molecular Probes, Eugene, OR, USA) and expressed as a relative median fluorescence intensity (MFI) ratio, calculated relative to the “rest” stage.
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