Anti mouse cd8

Manufactured by Thermo Fisher Scientific

Sourced in United States

Anti-mouse CD8 is a lab equipment product that detects the CD8 cell surface glycoprotein on mouse cells. It is used for the identification and characterization of CD8-positive cells in research applications.

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8 protocols using anti mouse cd8

Multiparameter Flow Cytometry Analysis

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Fluorescently conjugated antibodies were purchased from Ebioscience (anti-human CD3, anti-human CD8, anti-human PD1, anti-human CD25, anti-mouse CD3, anti-mouse CD8, anti-mouse PD1, anti-mouse CD25). Anti-human neuropilin-1 (FAB3870R) and anti-mouse neuropilin-1 (FAB566A) were purchased from R&D Systems. ITAg Tetramer - H-2 Kb OVA (SIINFEKL) conjugated to PE was purchased from MBL International Corporation, live/dead Fixable Viability Dye eFluor 450 and near infrared (Thermo Fisher). Celltrace violet cell proliferation kit was purchased from Thermo Fisher. Flow cytometric cell acquisition was done on a Canto II flow cytometer (BD Biosciences). Cell sorting was performed on a FACSAria II instrument (BD Biosciences). Flow cytometry data were analyzed using FlowJo software (TreeStar). Cells were gated on live cells (live/dead Fixable Viability Dye eFluor 450 negative).

Rossignol J., Belaid Z., Fouquet G., Guillem F., Rignault R., Milpied P., Renand A., Coman T., D’Aveni M., Dussiot M., Colin E., Levy J., Carvalho C., Goudin N., Cagnard N., Côté F., Babdor J., Bhukhai K., Polivka L., Bigorgne A.E., Halse H., Marabelle A., Mouraud S., Lepelletier Y., Maciel T.T., Rubio M.T., Heron D., Robert C., Girault I., Lebeherec D., Scoazec J.Y., Moura I., Condon L., Weimershaus M., Pages F., Davoust J., Gross D, & Hermine O. (2022). Neuropilin-1 cooperates with PD-1 in CD8+ T cells predicting outcomes in melanoma patients treated with anti-PD1. iScience, 25(6), 104353.

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Immunohistochemical Analysis of Immune Cells

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Brains from perfused mice were frozen in OCT (Fisher Scientific) and ten-micron thick sections were processed for immunohistochemistry. Briefly, sections were fixed in ice cold 95% ethanol for 15 min and washed in PBS several times. This was followed by washes in TBS with 0.1% Tween (TBST) and incubation for 10 min with 3% H₂O₂ to block endogenous peroxidase. After washing, blocking buffer was added for 1 h (10% normal goat serum in PBS). Primary antibody was added overnight at 4 °C: purified rat anti-mouse CD4, anti-mouse CD8 and anti-mouse F4/80 (all from eBiosciences), diluted 1:100 in PBS 2% normal goat serum. After washes in TBST, the biotinylated secondary antibody (anti-rat IgG, mouse absorbed, Vector) was added for 1 h, diluted 1:200 in PBS 2% normal goat serum. After washes in TBST, the Vectastain ABC reagent was used (Vector) following manufacturer’s instruction. Then, DAB (Sigma) was added as a substrate and, after incubation for 8 min in the dark and several washes in distilled water, sections were counterstained with Harris hematoxylin for 20 seconds, in lithium carbonate for 30 sec, washed in several changes of distilled water and mount with VectaMount AQ (Vector).

Casiraghi C., Citlali Márquez A., Shanina I, & Steven Horwitz M. (2015). Latent virus infection upregulates CD40 expression facilitating enhanced autoimmunity in a model of multiple sclerosis. Scientific Reports, 5, 13995.

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Immune Cell Isolation and Culture

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P. pavonica and J.rubens extracts were dissolved in phosphate-buffered saline (PBS) and kept at − 80 °C till further studies. Anti-mouse CD4, anti-mouse CD8, and anti-mouse CD335 (NKp46) monoclonal antibodies were purchased from eBioscience (San Diego, CA, USA). Heat-inactivated fetal bovine serum (FBS) (10% v/v), 2-mM l-glutamine, Penicillin/Streptomycin solution (100 IU/ml), 1-mM sodium pyruvate, non-essential amino acids are added to Roswell Park Memorial Institute medium 1640 (RPMI 1640). (Invitrogen, USA). Lonza, BioWhittaker, USA provided the ammonium-chloride-potassium (ACK) lysis buffer.

El-Sheekh M.M., Nassef M., Bases E., Shafay S.E, & El-shenody R. (2022). Antitumor immunity and therapeutic properties of marine seaweeds-derived extracts in the treatment of cancer. Cancer Cell International, 22, 267.

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Immunohistochemistry Staining of Tissue Samples

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Tissues for microscopic analysis were fixed overnight in 20% buffered formalin and transferred to 70% ethanol the next day. A Tissue-Tek VIP autoprocessor (Sakura, Torrance, CA) was used to process samples for paraffin-embedding. Tissue blocks were then sectioned to 4 μm, sections mounted on glass slides. Hematoxylin and Eosin (H&E) staining was performed at the University of California, Davis, Dept. of Pathology and Laboratory Medicine. Immune cells were stained with specific antibodies based on the manufacturer’s instructions. Anti-mouse CD8 (1:500 dilution; 14–0808, eBiosciences) was used as CD8 marker. Anti-mouse CD68 (1:700 dilution; PA1518, Boster) was used to stain macrophages. Anti-mouse OX40 antibody (1:2000 dilution; ab229021, abcam) was used to label the activated T cells. Anti-mouse CD206 (1:2000 dilution; ab64693, abcam) was used as a marker for M2 macrophages. Detection of the primary antibodies was performed using the appropriate isotype specific species secondary antibody with the Vectastain ABC Kit Elite Kit and a diaminobenzidine Peroxidase Substrate Kit (Vector Labs, Burlingame, CA) for amplification and visualization of signal. Stained slides were scanned on an AT2 Scanscope (Leica Biosystems), and digital images viewed using the Imagescope software.

Zhang H., Tang W.L., Kheirolomoom A., Fite B.Z., Wu B., Lau K., Baikoghli M., Raie M.N., Tumbale S.K., Foiret J., Ingham E.S., Mahakian L.M., Tam S.M., Cheng R.H., Borowsky A.D, & Ferrara K.W. (2020). Development of thermosensitive resiquimod-loaded liposomes for enhanced cancer immunotherapy. Journal of controlled release : official journal of the Controlled Release Society, 330, 1080-1094.

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Isolation and Characterization of Tumor-Infiltrating Immune Cells

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Isolation of cells from mouse tumors, cell-surface antigen staining, and flow cytometry were performed as described previously^{38 (link)}. Fluorescence-labelled anti-mouse CD45 (BioLegend), anti-mouse CD31 (BD Pharmingen), anti-mouse CD8 (eBioscience), and anti-mouse CD4 (eBioscience) were used. The stained cells were analysed and sorted by a FACSAria (BD Bioscience) and data were analysed using FlowJo Software (Treestar Software, San Carlos, California, USA). Dead cells were excluded by propidium iodide staining or by using the two-dimensional profile of the forward-versus-side scatter.

Hu L., Hayashi Y., Kidoya H, & Takakura N. (2021). Endothelial cell-derived Apelin inhibits tumor growth by altering immune cell localization. Scientific Reports, 11, 14047.

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Immune Cell Profiling by Flow Cytometry

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Cells were subjected to fluorescence-activated cell sorting (FACS) analysis with specific antibodies. The antibodies used for cell surface staining were: anti-mouse CD45 and anti-mouse CD206 (TONBO); anti-mouse CD11b, anti-mouse CD11c, anti-mouse CD8, anti-mouse F4/80, and anti-mouse CD209a (eBioscience); anti-mouse CD4, anti-mouse CD3, anti-mouse Ly6C, and anti-mouse Ly6G (Biolegend). Data were acquired with an LSRII flow cytometer (BD Biosciences) and analyzed with FlowJo V10.6 (Tree Star Inc.).

Wu C.J., Pan K.F., Chen J.Q., Tao Y.C., Liu Y.C., Chen B.R., Hsu C., Wang M.Y., Sheu B.C., Hsiao M., Hua K.T, & Wei L.H. (2024). Loss of LECT2 promotes ovarian cancer progression by inducing cancer invasiveness and facilitating an immunosuppressive environment. Oncogene, 43(7), 511-523.

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Larval Dissection and Immunohistochemistry

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Dissection, staining, and mounting of wandering third instar larvae was performed as previously described (Sulkowski et al., 2011 (link)). For immunohistochemistry (IHC), larvae were dissected in PBS, pinned on sylgard plates and fixed in 4% paraformaldehyde for 20 minutes. Next, larvae were washed five times in 1X PBT (PBS + 0.3% Triton X-100). Cuticle filets were blocked in 5% normal donkey serum (Jackson Laboratories, West Grove, PA) for at least 30 minutes at room temperature followed by incubation with respective primary antibodies. Antibody dilutions used were as follows: rabbit anti-Bdwf (1:200), mouse anti-CD8 (1:100) (Invitrogen), rabbit RpS6-(1:100) (Cell Signaling Technology), and mouse anti-Cut (2B10; 1:50) (Developmental Studies Hybridoma Bank). Donkey anti-rabbit and donkey anti-mouse secondary antibodies were used at 1:200 (Jackson Immunoresearch). Filets were incubated in glycerol for 5 minutes, followed by being directly mounted onto coverslips with either a drop of glycerol, or aqueous fluoromount. The slides were then imaged on a Nikon C1 Plus confocal microscope or Zeiss LSM 780.

Bhattacharjee S., Iyer E.P., Iyer S.C., Nanda S., Rubaharan M., Ascoli G.A, & Cox D.N. (2023). The Zinc-BED transcription factor Bedwarfed promotes proportional dendritic growth and branching through transcriptional and translational regulation in Drosophila. bioRxiv.

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Larval Dissection and Immunostaining

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Dissection, staining, and mounting of wandering third-instar larvae were performed as previously described [19 (link)]. For immunohistochemistry (IHC), larvae were dissected in PBS, pinned on Sylgard plates, and fixed in 4% paraformaldehyde for 20 minutes. Next, larvae were washed five times in 1× PBT (PBS + 0.3% Triton X-100). Cuticle filets were blocked for at least 30 minutes at room temperature in 5% normal donkey serum (Jackson Laboratories, West Grove, PA, USA), followed by incubation with respective primary antibodies. Antibody dilutions used were as follows: rabbit anti-Bdwf (1:200), mouse anti-CD8 (1:100) (Invitrogen), rabbit RpS6-(1:100) (Cell Signaling Technology, Danvers, MA, USA), and mouse anti-Cut (2B10; 1:50) (Developmental Studies Hybridoma Bank, Houston, TX, USA). Donkey anti-rabbit and donkey anti-mouse secondary antibodies were used at 1:200 (Jackson Immunoresearch, West Grove, PA, USA). Filets were incubated in glycerol for 5 minutes, followed by being directly mounted onto coverslips with either a drop of glycerol or an aqueous fluoromount. The slides were then imaged on a Nikon C1 Plus confocal microscope or a Zeiss LSM 780.

Bhattacharjee S., Iyer E.P., Iyer S.C., Nanda S., Rubaharan M., Ascoli G.A, & Cox D.N. (2023). The Zinc-BED Transcription Factor Bedwarfed Promotes Proportional Dendritic Growth and Branching through Transcriptional and Translational Regulation in Drosophila. International Journal of Molecular Sciences, 24(7), 6344.

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