Pe conjugated anti mouse cd8

Manufactured by Thermo Fisher Scientific

Sourced in United States

The PE-conjugated anti-mouse CD8 is a lab equipment product that is used for the detection and analysis of CD8-positive cells in mouse samples. It functions as a fluorescent-labeled antibody that specifically binds to the CD8 receptor on the surface of mouse T cells, allowing for their identification and quantification through flow cytometry or other immunoassay techniques.

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8 protocols using pe conjugated anti mouse cd8

Comprehensive Immunophenotyping of T-cells and DCs

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T-cell suspensions were isolated from the spleen and lymph nodes, and were stained with antibodies against the following cell surface antigens: APC-conjugated anti-mouse CD3, FITC-conjugated anti-mouse CD4, PE-conjugated anti-mouse CD8, PE-conjugated anti-mouse Gr-1, APC-conjugated anti-mouse CD11b, PE-conjugated anti-mouse TCR (eBioscience), PE-conjugated anti-mouse PD-1, and PE-conjugated anti-mouse CD152 (CTLA-4; BD Bioscience, San Jose, CA, USA). Tregs were detected from the spleen and lymph nodes using a Mouse Regulatory T-Cell Staining Kit (eBioscience) according to the manufacturer's instructions. DCs were isolated from the bone marrow and cells were incubated with FITC-conjugated anti-CD86, APC-conjugated anti-CD11C, PE-conjugated anti-MHC-II, FITC-dextran, PE-conjugated anti-CCR7, and anti-PD-L1 (eBioscience). The cells were analyzed by FACS, and the acquired data was performed with FlowJo Software, version 9.1 (Tree Star, San Carlos, CA, USA).

Liu X., Zhou Z., Cheng Q., Wang H., Cao H., Xu Q., Tuo Y., Jiang L., Zou Y., Ren H, & Xiang M. (2017). Acceleration of pancreatic tumorigenesis under immunosuppressive microenvironment induced by Reg3g overexpression. Cell Death & Disease, 8(9), e3033-.

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Spleen Cell Immunophenotyping by Flow Cytometry

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The mice were humanely sacrificed, and the spleens were removed. The spleens were triturated, and the red blood cells were removed using RBC Lysis Buffer (Solarbio) according to the kit instructions. After centrifuging at 500 g for 10 min at 4°C, the supernatants were discarded, and cells were resuspended and washed twice with Staining buffer (eBioscience) to obtain single-cell suspensions. Then, cells were stained with different combinations of flow cytometry antibodies, including APC-conjugated anti-mouse CD3 (eBioscience, San Diego, United States), FITC-conjugated anti-mouse CD4 (BioLegend, San Diego, United States), PE-conjugated anti-mouse CD8 (eBioscience) APC-conjugated anti-mouse B220 (BioLegend), Pacific Blue-conjugated anti-mouse GL-7 (BioLegend), PE-conjugated anti-mouse CD95 (BioLegend), PE-conjugated anti-mouse PD-1 (BioLegend), and Brilliant Violet 421-conjugated anti-mouse CXCR5 (BioLegend). After staining, cells were washed with Staining buffer and dispersed in 500 mL of Staining buffer. Analysis was performed using a Mona CytoFLEX flow cytometer (BeckmanCoulter LifeSciences, Brea, United States).

Li S., Huang J., Wang K., Liu Y., Guo Y., Li X., Wu J., Sun P., Wang Y., Zhu L, & Wang H. (2023). A bioconjugate vaccine against Brucella abortus produced by engineered Escherichia coli. Frontiers in Bioengineering and Biotechnology, 11, 1121074.

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Characterization of Thymocyte Subsets

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Total thymocytes were isolated from the thymus in HSC buffer (HBSS with Ca2⁺ and Mg2⁺, 5% fetal bovine serum, 2 mM EDTA). Red blood cells (RBC) were lysed using ACK lysing buffer (Lonza). 1 x 10⁶ live thymocytes were blocked with a rat anti-mouse CD16/32 antibody (BD Pharmingen) and stained with a combination of the antibodies including PE-Cy5 conjugated anti-mouse CD4 (clone: GK1.5, eBioscience), PE conjugated anti-mouse CD8 (clone: 53-6.7, eBioscience), APC conjugated anti-mouse CD44 (clone: IM7, eBioscience), BV421 conjugated anti-mouse CD25 antibodies (clone: PC61, BioLegend). FITC conjugated anti-mouse CD45.2 (clone: 104, eBioscience), and APC-eFluor780 conjugated anti-mouse CD45.1 (clone: A20, e eBioscience). All antibodies were diluted 1:400. Data were collected from at least 100,000 single cells by FACSCanto (BD Pharmingen) and analyzed by FlowJo (Tree Star, Inc) without knowledge of the genotype or treatment by a single observer (C-LL).

Lee C.L., Brock K.D., Hasapis S., Zhang D., Sibley A.B., Qin X., Gresham J.S., Caraballo I., Luo L., Daniel A.R., Hilton M.J., Owzar K, & Kirsch D.G. (2021). Whole exome sequencing of radiation-induced thymic lymphoma in mouse models identifies Notch1 activation as a driver of p53 wild-type lymphoma. Cancer research, 81(14), 3777-3790.

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Single-cell Analysis of Wound-Derived T Cells

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Seven days after wounding, 18 skin wounds from three mice were harvested using a 5mm punch-biopsy instrument. A single-cell suspension was obtained using a tissue dispersion method (18 (link)). For control cells, spleens from unwounded mice were removed, minced, and passed through a 70-mm cell strainer to make single cell suspensions. Red blood cells were lysed using a RBC lysis buffer (eBioscience, San Diego, CA). Cell suspensions from both wound and spleen were then incubated with FITC conjugated rat anti-mouse CD4, PE-conjugated anti-mouse CD8, and APC-conjugated anti-mouse CD11a (eBioscience) for 30min on ice. All antibodies were used at the concentration of 0.5µg/ml. After washing, the CD4&CD11a or CD8&CD11a double stained cells were analyzed and sorted using a Moflo high speed cell sorter (Dako cytomation, Glostrup, Denmark). Dead cells were gated out by 7-AAD staining. 3,000–10,000 sorted CD8+ and CD4+ cells were obtained from the wounds. Cells-Ct kit (Invitrogen) was used to prepare cDNA for cytokine real time PCR.

Chen L., Mehta N.D., Zhao Y, & DiPietro L.A. (2014). Absence of CD4 or CD8 lymphocytes changes infiltration of inflammatory cells and profiles of cytokine expression in skin wounds, but does not impair healing. Experimental dermatology, 23(3), 189-194.

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Flow Cytometry Analysis of Tumor-Infiltrating Lymphocytes

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For the flow cytometry analysis, CD8⁺ T cells were stained with APC-conjugated anti-mouse PD-1 Ab and FITC-conjugated anti-mouse IFN-γ Ab (eBioscience, CA, USA). To evaluate the tumor-infiltrating lymphocytes (TILs), a single-cell suspension from the implanted tumors was stained with the following Abs: APC-conjugated anti-mouse CD3, PE-conjugated anti-mouse CD8 and FITC-conjugated anti-mouse CD4 (eBioscience, CA, USA). Flow cytometry was performed on a BD Accuri C6 flow cytometer (BD Bioscience) and analyzed with BD Accuri C6 software.

Shi J., Chen C., Ju R., Wang Q., Li J., Guo L., Ye C, & Zhang D. (2019). Carboxyamidotriazole combined with IDO1-Kyn-AhR pathway inhibitors profoundly enhances cancer immunotherapy. Journal for Immunotherapy of Cancer, 7, 246.

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Thymocyte Isolation and Characterization

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Total thymocytes were isolated from the thymus according to methods described elsewhere (16 (link)). A total of 1 × 10⁶ live thymocytes were blocked with a rat anti-mouse CD16/32 antibody (BD Pharmingene, San Diego, CA) and stained with a combination of the antibodies including PE-Cy5 conjugated anti-mouse CD4 (clone: GK1.5), PE conjugated anti-mouse CD8 (clone: 53–6.7), APC conjugated anti-mouse CD44 (clone: IM7), FITC conjugated anti-mouse CD45.2 (clone: 104), and APC-eFluor780 conjugated anti-mouse CD45.1 (clone: A20), all acquired from eBioscience^™ Inc. (San Diego, CA), as well as BV421 conjugated anti-mouse CD25 antibodies (clone: PC61; BioLegend^® Inc., San Diego, CA). All antibodies were diluted 1:400. Data were collected from at least 100,000 single cells using FACSCanto^™ (BD Pharmingen) and analyzed using FlowJo (Tree Star Inc., Ashland, OR) without knowledge of the genotype or treatment by a single observer (C-LL).

Hasapis S., Caraballo I, & Lee C.L. (2021). Transplantation of Unirradiated Bone Marrow Cells after Total-Body Irradiation Prevents the Development of Thymic Lymphoma in Mice through Niche Competition. Radiation research, 195(3), 301-306.

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Thymocyte Phenotyping by Flow Cytometry

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Total thymocytes were isolated from the thymus in HSC buffer (HBSS with Ca2⁺ and Mg2⁺, 5% fetal bovine serum, 2 mM EDTA). Red blood cells (RBC) were lysed using ACK lysing buffer (Lonza). Total number of thymocytes was counted with a Coulter counter (Beckman Coulter). 1 × 10⁶ thymocytes were blocked with a rat anti-mouse CD16/32 antibody (BD Pharmingen) and stained with PE-Cy5 conjugated anti-mouse CD4 (clone: GK1.5), PE conjugated anti-mouse CD8 (clone: 53-6.7), APC conjugated anti-mouse CD44 (clone: IM7), FITC conjugated CD25 (clone: PC61.5) and APC-eFluor780 conjugated anti-mouse cKit (clone: 2B8) antibodies (eBioscience). All antibodies were diluted 1:400. Dead cells were excluded by staining with Calcein Blue AM (Life technologies). Data were collected from 250,000 single cells by FACSCanto (BD Pharmingen) and analyzed by FlowJo (Tree Star, Inc) without knowledge of the genotype or treatment by a single observer (CLL).

Lee C.L., Castle K.D., Moding E.J., Blum J.M., Williams N., Luo L., Ma Y., Borst L.B., Kim Y, & Kirsch D.G. (2015). Acute DNA damage activates the tumour suppressor p53 to promote radiation-induced lymphoma. Nature Communications, 6, 8477.

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T Cell Activation Signaling Dynamics

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Purified T cells were plated 0.5 × 10⁶ per well and allowed to settle, then 1 µg/mL biotin labeled anti-CD3 and anti-CD28 (Thermo Fisher Scientific, Cat#11456D) added for 5 min at 4 °C and crosslinked with strepatvidin (20 µg/mL). At 1,5 and 15 min time points 4% PFA was added to cell mixture and fixed at 27 °C for 15 min. Cells were washed with PBS, permeabilized with 100% ice-cold methanol and placed at −20 °C for 10 min. Permeabilized cells were incubated in blocking buffer (2% FCS/PBS + Na₃VO₄ + NaF + NaCl) at RT for 30 min. Cells were then stained with BV605-conjugated anti-mouse CD4 (Biolegend, Cat#100547), PE-conjugated anti-mouse CD8 (eBioscience, Cat#12-0081-82) and 1:150 diluted anti-phospho-Akt (S473) (Cell Signaling Technology, Cat#9271) antibody for 30 min at RT. Cells were washed twice with PBS and stained with 1:4000 Alexa Fluor 647 Goat anti-rabbit IgG (Life Technologies, Cat# A21245) at RT for 30 min. Cells were washed twice with PBS and resuspended in 200 µL PBS for cytofluorimetric analysis.

Bonacina F., Coe D., Wang G., Longhi M.P., Baragetti A., Moregola A., Garlaschelli K., Uboldi P., Pellegatta F., Grigore L., Da Dalt L., Annoni A., Gregori S., Xiao Q., Caruso D., Mitro N., Catapano A.L., Marelli-Berg F.M, & Norata G.D. (2018). Myeloid apolipoprotein E controls dendritic cell antigen presentation and T cell activation. Nature Communications, 9, 3083.

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