Anti cd8 apc

Manufactured by BioLegend

Sourced in United States, Germany

Anti-CD8-APC is a fluorochrome-conjugated monoclonal antibody that binds to the CD8 surface antigen. CD8 is a co-receptor expressed on cytotoxic T cells and a subset of natural killer cells. The APC fluorochrome allows for detection and quantification of CD8+ cells using flow cytometry.

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57 protocols using anti cd8 apc

Comprehensive Immune Profiling by Flow Cytometry

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For in-vitro studies, the following flow cytometry panel was used in conjunction with CTV and viability dye: anti-CD3 Alexa700, anti-CD4 PE-Cy7, anti-CD8 APC, and anti-CD44 PE, all at a 1:200 dilution (Biolegend). For in-vivo and ex-vivo studies, the following flow cytometry panel was used: anti-CD3 PE-Cy7, anti-CD4 Pacific Blue, anti-CD8 APC, anti-CD44 PE, anti-CD62L APC-Cy7, and anti-CD25 Alexa 700, all at a 1:200 dilution, except anti-CD25 at a 1:50 dilution (Biolegend). Endogenous Foxp3 expression was detected either via eGFP of eYFP fluorescence, which is compatible with all panels but not when used with BODIPY-FL dye (493/503) or 2-NBDG (465/540). In those assays, Foxp3-based uptake of FA or Glucose was not performed, but all other cellular subsets described were tested. All acquisition was performed using a BD Fortessa flow cytometer (BD, MD, USA) at the Northwestern University Comprehensive Flow Cytometry core.

Muroski M.E., Miska J., Chang A.L., Zhang P., Rashidi A., Moore H., Lopez-Rosas A., Han Y, & Lesniak M.S. (2017). Fatty Acid Uptake in T Cell Subsets Using a Quantum Dot Fatty Acid Conjugate. Scientific Reports, 7, 5790.

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Skin Immune Profiling in Murine Model

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Mice (n = 9) were sacrificed on Day 3 and had their skin harvested for histological and flow cytometry analysis. Murine back skin sections were digested to assess immune cell populations as previously described^{34 (link)}. Briefly, precut skin sections were digested with Collagenase P (11213857001; Sigma-Aldrich), DNAse I (9003-98-9; Worthington) and Dispase II (D4693-1G; Sigma-Aldrich) in DMEM (Wisent Bio Products) for 1 h in 37°C. Cells were then filtered through 40 μm cell strainers before being stained for flow cytometry. Next, Fc receptors on cells were blocked with Mouse TruStain FcX (101319; BioLegend) and stained with cocktail combinations of: APC/Cy7 anti-CD45 (103116; Biolegend), PE/Cy5 anti-CD3 (100310; BioLegend), APC anti-CD8, PE/Cy7 anti-CD4, Alexa Fluora 700 anti-CD11b (101222; Biolegend), FITC anti-NKp46 (137605; Biolegend), PE anti-TIGIT, PerCP Cy5.5 anti-CD226, FITC anti-F4/80 (123108; Biolegend), APC anti-Ly6C (128015; Biolegend), and PE/Cy5 anti-CD11c (117316; Biolegend).
Additionally, skin samples were sectioned and stained with H&E for measuring epidermal thickness. Histology section images were captured, and epidermal thickness was measured using S-EYE 1.4 software. The epidermal thickness was quantified by measuring thickness in at least three distinct locations on each tissue section.

Saha S., Sparkes A., Matus E.I., Lee P, & Gariépy J. (2023). The IgV domain of the poliovirus receptor alone is immunosuppressive and binds to its receptors with comparable affinity. Scientific Reports, 13, 4609.

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Phenotypic Characterization of Immune Cells

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After harvesting and washing with PBS, cells were stained with PE anti-mouse CD197 (clone 4B12, eBioscience), APC anti-mouse CD86 (clone GL-1, Biolegend), PE anti-MMR (anti-CD206) (clone FAB2535P, R&D), PE anti-PDL-1 (clone 10F.9G2, Biolegend) or IgG control antibody (Biolegend, R&D) for 30 min at room temperature. After washing with 1xPBS, cells were immediately analyzed using a Caliber flow cytometer (Becton and Dickinson). Percentage of gated cells was calculated and analyzed using CellQuest ProTM software (Becton and Dickinson).
MLR cultures were harvested and stained with APC anti-CD8 (Biolegend) and PE anti-CD4 (Biolegend). CFSE staining (Invitrogen, Molecular Probes) was performed in CD8⁺ or CD4⁺ T cells subsets following manufacturers’ protocol at 2–3 days after co-culture was started.

Seth P., Csizmadia E., Hedblom A., Vuerich M., Xie H., Li M., Longhi M.S, & Wegiel B. (2017). Deletion of lactate dehydrogenase-A in myeloid cells triggers antitumor immunity. Cancer research, 77(13), 3632-3643.

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Multiparametric flow cytometry panel

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Cells were suspended in MACS buffer and incubated with anti-CD16/32 mAb (BioLegend, 101310) for Fc receptor blocking. Cells were stained with the following fluorochrome-conjugated antibodies: PerCP/Cy5.5 anti-CD45 (BioLegend, 103131), APC anti-CD45 (BioLegend, 103111), PE/Cy7 anti-CD11b (Biolegend, 101215), FITC anti-CD3 (BD Biosciences, 553061), PE anti-CD4 (BD Biosciences, 553049), APC anti-CD8 (BioLegend, 100712), PerCP/Cy5.5 anti-CD11c (BioLegend, 117328), PE/Cy7 anti-MHC II (BioLegend, 107630), FITC anti-CD11b (BioLegend, 101206), V421 anti-CD64 (BioLegend, 139309), PE anti-Ly6G (BioLegend, 127607), APC anti-B220 (BioLegend, 103211), and PE anti-NK1.1 (BD Biosciences, 553165). Stained cells were analyzed by BD FACSVerse (BD bioscience). Data analysis was performed using FlowJo software (Treestar).

Horiguchi H., Kadomatsu T., Yamashita T., Yumoto S., Terada K., Sato M., Morinaga J., Miyata K, & Oike Y. (2023). ANGPTL2 promotes immune checkpoint inhibitor-related murine autoimmune myocarditis. Communications Biology, 6, 965.

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Multiparametric Flow Cytometry of Immune Cells

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Mouse isolated T cells were analyzed using flow cytometry (CytoFLEX, Beckman Coulter, Lakeview Indianapolis, IA) for expression of GFP following pMIGII viral transduction or by staining with Alexa Fluor 647 anti-mouse CD64 (FcγRI) (X54–5/7.1, BioLegend), Brilliant Violet 421 or APC or PE anti-human CD64 (10.1, BioLegend), FITC-anti-human CD3 (UCH1, BioLegend), Brilliant Violet 421-anti-CD4 (RPA-T4), APC–anti-CD8 (RPA-T8), PE-anti-γδ-TCR (BioLegend, #331209), anti–PD-1 (BioLegend, #329924), anti–LAG-3 (BioLegend, #369208), anti-Tim3 (BioLegend, 345016). Intracellular staining of FcRγ was performed using Milli mark anti-FcεRI γ subunit -FITC (Merck, FCABS400F). Datasets were analyzed using FlowJo software (Tree Star, version 10.7.2).

Rasoulouniriana D., Santana-Magal N., Gutwillig A., Farhat-Younis L., Tal L., Amar S., Milyavsky M., Muddineni S.S., Solomon N., Shpilt H., Dotan S., Pilpel N., Waskow C., Feinmesser M., Rider P, & Carmi Y. (2023). T Cells Expressing a Modified FcγRI Exert Antibody-Dependent Cytotoxicity and Overcome the Limitations of CAR T-cell Therapy against Solid Tumors. Cancer Immunology Research, 11(6), 792-809.

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Phenotypic Analysis of Immune Cell Subsets

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Cells were suspended in MACS buffer and incubated with anti-CD16/32 mAb (BioLegend, 101310) for Fc receptor blocking. Cells were stained with the following fluorochrome-conjugated antibodies: PerCP/Cy5.5 anti-CD45 (BioLegend, 103131), PE anti-CD45 (BioLegend, 109807), FITC anti-CD8 (MBL, D271-4), APC anti-CD8 (BioLegend, 100712), PE anti-GzmB (BD, 561142), PerCP/Cy5.5 anti-CD11c (BioLegend, 117328), PE/Cy7 anti-MHC II (BioLegend, 107630), PE anti-CD86 (BioLegend, 105008), APC anti-CD80 (BioLegend, 104713), FITC anti-CD40 (BioLegend, 102905), PE anti-ITGα5 (BioLegend, 103805), anti-ITGα4 (BioLegend, 103607), anti-ITGαv (BioLegend, 104105), anti-ITGβ1 (BioLegend, 102207), anti-ITGβ2 (BioLegend, 101407), anti-ITGβ3 (BioLegend, 104307), and anti-PIR-A/B (BD, 550349). For analysis of OVA-specific CD8⁺ T cells, cells were incubated with PE-conjugated H-2K^b-SIINFEKL-tetramer (MBL), according to the manufacturer's instructions. Stained cells were analyzed by BD FACSVerse (BD bioscience). Data analysis was performed using FlowJo software (Treestar).

Horiguchi H., Kadomatsu T., Kurahashi R., Hara C., Miyata K., Baba M., Osumi H., Terada K., Araki K., Takai T., Kamba T., Linehan W.M., Moroishi T, & Oike Y. (2019). Dual functions of angiopoietin-like protein 2 signaling in tumor progression and anti-tumor immunity. Genes & Development, 33(23-24), 1641-1656.

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Antigen-Specific T-Cell Proliferation Assay

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Mice were sacrificed 35 days postimmunization. Mononuclear cells (MNC) from lymph nodes and spleen were passed through a 40-mm nylon mesh, enriched with a Ficoll gradient centrifugation and adjusted to a concentration of 2 × 10⁶ cells/ml. Antigen-induced T-cell proliferation was measured per the following procedures. MNC were stained with CFDA-SE [5 µM, 8 min, room temperature (RT)] and cultured in 96-well plates (2 × 10⁵ cells/well). Antigens (MBP, LMP1, OVA) were added to the culture at 1:1 ratio to the final concentration of 15 µM. Three days later cells were harvested and stained with APC-anti-CD8 and PerCP-Cy5.5-anti-CD4 mAbs (Biolegend, San Diego, CA, USA). Proliferation of CD4⁺ and CD8⁺ cells were assessed by flow cytometry based on CFSE dilution. All experiments were performed on BD FACS Canto II (BD Biosciences, San Jose, CA, USA). Unstimulated cells and cells stimulated with immobilized anti-CD3 mAb were used as negative and positive controls, respectively.

Lomakin Y., Arapidi G.P., Chernov A., Ziganshin R., Tcyganov E., Lyadova I., Butenko I.O., Osetrova M., Ponomarenko N., Telegin G., Govorun V.M., Gabibov A, & Belogurov A J.r. (2017). Exposure to the Epstein–Barr Viral Antigen Latent Membrane Protein 1 Induces Myelin-Reactive Antibodies In Vivo. Frontiers in Immunology, 8, 777.

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Transfection and Bioluminescence Imaging

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HEK293 (1 × 10⁶) cells were resuspended in 10 μL of T buffer and transfected with 2 μg each of eGFP and luciferase plasmids in 10 μL tips using a Neon transfection system (Life Technologies, Foster City, CA). Fluorescence or luminescence was monitored over a 24 h period. eGFP-positive cells were detected using an Olympus IX71 inverted fluorescence microscope and counted in 10 random fields at 100× magnification. Bioluminescent signals from luciferase transfected cells were monitored using the IVIS Lumina (Perkin Elmer, Waltham, MA, USA). To assess localization of luciferase transfected cells in vivo, after i.p. injections of 0.1 mL of 15 mg/mL D-luciferin, bioluminescence signals of vaccinated mice were evaluated using the IVIS Lumina. Regions of interest were identified around the injection sites and quantified as total photon counts using Living Image^™ software. The values were expressed as average of all vaccinated mice for every treatment group. Flow cytometric detection of CD8⁺ T cells expressing intracellular perforin were performed as previously described [18 (link)]. APC-anti-CD8 and PE-anti-perforin (BioLegend) were used at a final dilution of 1:500 and 1:200, respectively.

Bertino P., Urschitz J., Hoffmann F.W., You B.R., Rose A.H., Park W.H., Moisyadi S, & Hoffmann P.R. (2014). Vaccination with a piggyBac plasmid with transgene integration potential leads to sustained antigen expression and CD8+ T cell responses. Vaccine, 32(15), 1670-1677.

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Antibody-based Western Blot and Flow Cytometry

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Antibodies for Western blots included anti-MEK1, anti-ERK1/2, and anti-p-ERK1/2 (catalog no.: 12671S, 1:1000 dilution; catalog no.: 9107S, 1:1000 dilution; catalog no.: 4370T, 1:500 dilution; Cell Signaling); anti-FLAG (catalog no.: F1804; 1:1000 dilution; Millipore Sigma), anti-GAPDH (catalog no.: ab9485; 1:1000 dilution; Abcam), anti-p-Elk1 (catalog no.: 43004; 1:1000 dilution; QED Bioscience), anti-Elk1 and anti-β-actin (catalog no.: SC-365876, 1:1000 dilution and SC-517582, 1:2000 dilution, respectively; Santa Cruz). BioLegend antibodies for flow cytometry used at manufacturer's recommended concentrations included FITC-anti-CD3 (catalog no.: 100306), APC-anti-CD8 (catalog no.: 100712), and PE-anti-CD45.2 (catalog no.: 109808). Dead cells were detected with BV421 viable dye (Invitrogen). Mimic Dharmacon miRIDIAN reagents for miR-15a and miR-16 (catalog nos.: CTM-535609 and CTM-535610) as well as negative control mimic (catalog no.: CTM-535611) were purchased with 5′-fluorescein and 3′-cholesterol modifications. DOX was purchased from Sigma.

Urena F., Ma C., Hoffmann F.W., Nunes L.G., Urschitz J., Moisyadi S., Khadka V.S., Deng Y, & Hoffmann P.R. (2022). T-cell activation decreases miRNA-15a/16 levels to promote MEK1–ERK1/2–Elk1 signaling and proliferative capacity. The Journal of Biological Chemistry, 298(3), 101634.

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Flow Cytometry Immune Cell Profiling

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Cells were resuspended in 100 μl FCB, blocked with 0.5 μl TruStainFcX (anti-mouse CD16/32 FcR block, Biolegend) for 5 min at room temperature, and stained for 30 min at 4°C with: eFluor605NC-anti-CD45 (1:500, Biolegend), FITC-anti-CD4 (1:200, eBioscience), APC-anti-CD8 (1:200, Biolegend), PE-anti-B220 (1:200, Biolegend), APC-eFluor780-anti-MFICII (1:200, eBioscience), PE-Cy7-anti-CD11b (1:200, eBioscience) and PerCP-Cy5.5-anti-Ly6G (1:200, Biolegend). Cells were then fixed in 1% PFA for 1 h. Finally, cells were resuspended in 500 μl FCB, labelled with DAPI to exclude cell debris and analyzed with a CytoFLEX S flow cytometer (Beckman-Coulter) equipped with CytExpert software (Beckman-Coulter).

Madsen P.M., Desu H.L., Vaccari J.P., Florimon Y., Ellman D.G., Keane R.W., Clausen B.H., Lambertsen K.L, & Brambilla R. (2019). Oligodendrocytes modulate the immune-inflammatory response in EAE via TNFR2 signaling. Brain, behavior, and immunity, 84, 132-146.

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