α cd8

Manufactured by Thermo Fisher Scientific

Sourced in United States

The α-CD8 is a laboratory equipment used for the detection and analysis of CD8+ T cells. It provides a reliable and precise method for the identification and quantification of CD8-positive cells in various biological samples.

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Lab products found in correlation

10 protocols using α cd8

4T1 Cell-mediated Cytotoxicity Assay

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4T1 cell line variants were cultured in the absence or presence of 100 ng/mL recombinant IFN-gamma in 37 °C 5% CO₂ for 24 h^{38 (link)}. About 5 × 10⁴ treated cells were washed twice with PBS and incubated with 5 × 10³ 4T1-specific CTL in the presence of 10 µg/mL Brefeldin A in 37 °C 5% CO₂ for 5 h. For anti-PD1 experiments: 4T1-specific CD8 + T cells were incubated in the presence of 10 µg/mL anti-PD1 (clone: RMP1-14, BioXcell, cat#BE0146) or isotype control (clone: 2A3, BioXcell, cat#BE0089) for 30 min, RT. Samples were then stained with cell surface antibodies for 20 min, 4 °C (α-CD8, clone: 53-6.7, 1:300, eBioscience), washed with PBS, fixed with 1% paraformaldehyde (15 min, RT, in the dark), permeabilized and stained for intracellular proteins (α-IFN-gamma, clone: XMG1.2, 1:300, eBioscience) in the presence of 0.4% Saponin for 30 min, 4 °C, and analysed on a FACSymphony A5 (BD).

Baldwin L.A., Bartonicek N., Yang J., Wu S.Z., Deng N., Roden D.L., Chan C.L., Al-Eryani G., Zanker D.J., Parker B.S., Swarbrick A, & Junankar S. (2022). DNA barcoding reveals ongoing immunoediting of clonal cancer populations during metastatic progression and immunotherapy response. Nature Communications, 13, 6539.

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Measuring CD8+ T Cell Response to HA Peptide

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Following isolation, lymphocytes were incubated at 37°C for 5 hours with or without the x31 derived Haemagglutinin (HA) (195–209) (YVQASGRVTVSTRRS) peptide (ANASPEC INC, Fremont, CA) in the presence of GolgiStop (BD Pharmingen, San Diego, CA). 1×10⁶ naïve splenocytes were added to the lymphocyte populations isolated from the BAL as an antigen presenting population. Following the stimulation period the cells were extracellularly stained with αCD8, αCD4 and αCD44 antibodies (eBioscience, San Diego, CA) for 20 min at 4°C, fixed in 2% paraformaldehyde overnight, permeabilized using Perm/Wash Buffer (BD Biosciences, San Diego, CA) and intracellularly stained using FITC conjugated αIFN-γ (BD Pharmingen, San Diego, CA) for 30 min at 4°C. Samples were analyzed by flow cytometry as described above.

Shane H.L, & Klonowski K.D. (2014). A direct and non-redundant role for thymic stromal lymphopoietin on anti-viral CD8 T cell responses in the respiratory mucosa. Journal of immunology (Baltimore, Md. : 1950), 192(5), 2261-2270.

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Detailed Workflow for Murine T Cell Analysis

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Isolated cells were stained with αCD4, αCD8, αCD3, αTCRβ, αCD44, αCD62L, αFoxP3, αIL-17 and αIFN-γ (eBioscience and Tombo). For FoxP3 and cytokine staining, cells were stained according to the manufacturer’s protocol form FoxP3 staining kit (Affimetrix/eBioscience). For cytokine staining, cells were stimulated with 50 ng/ml PMA and 1 μg/ml ionomycin in the presence of GolgiPlug (BD Bioscience) for 4 hours at 37°C and stained for surface markers followed by fixation/permeabilization and staining for cytokines. Stained cells were acquired with LSRII or LSR Fortessa cytometers; data was analyzed using FlowJo (Treestar) (BD Bioscience). For sorting of Treg cells, CD4+ T cells were enriched by using mouse CD4+ T cell isolation kit (Miltenyi) or Magnisort mouse CD4+ T cell enrichment kit (Affimetrix/eBioscience) and then cells were sorted by FACSAria (BD Bioscience).

Oh H., Grinberg-Bleyer Y., Liao W., Maloney D., Wang P., Wu Z., Wang J., Bhatt D.M., Heise N., Schmid R.M., Hayden M.S., Klein U., Rabadan R, & Ghosh S. (2017). An NF-κB transcription factor-dependent, lineage specific transcriptional program promotes regulatory T cell identity and function. Immunity, 47(3), 450-465.e5.

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PLGA-Based Antigen Delivery System

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Poly (D, L-lactic-co-glycolic acid) (75:25 PLGA, Resomer® RG 752H, M_W 4A) was purchased from Lakeshore Biomaterials (Birmingham, AL, USA). Ovalbumin (OVA_257-264) and dimethyl-dioctadecyl-ammonium bromide (DDAB) were obtained from Sigma (St, Louis, MO, USA). Methylene chloride (AR grade) and absolute ethyl alcohol (AR grade) were purchased from Beijing Chemical Reagent Company (Beijing, China). Concanavalin A, Roswell Park Memorial Institute (RPMI) 1640 medium, Dulbecco's modified Eagle medium (DMEM) medium, and fetal bovine serum (FBS) were supplied by Gibco (Grand Island, NY, USA). Alexa 635-phalloidin and Lysol-Tracker probes were purchased from Invitrogen (Grand Island, NY, USA). FITC was obtained from Sigma-Aldrich. Fluorochrome-labeled α-MHCI, α-MHC II, α-CD86, α-CD80, α-CD11c, α-CD69, α-CD44, α-CD62L, α-CD4, α-CD8 and α-CD19 antibodies were supplied by eBioscience (San Diego, CA, USA). Recombinant mouse GM-CSF and IL-4 were obtained from Peprotech (Rocky Hill, NJ, USA). Mouse cytokine ELISA kits were purchased from eBioscience. OVA derived (H-2 Kb, SIINFEKL) specific MHC I pentamers was purchased from ProImmune (Oxford, UK). p38 MAPK inhibitor SB203580 was purchased from Cell Signaling Technology (Danvers, MA, USA).

Han S., Ma W., Jiang D., Sutherlin L., Zhang J., Lu Y., Huo N., Chen Z., Engle J.W., Wang Y., Xu X., Kang L., Cai W, & Wang L. (2021). Intracellular signaling pathway in dendritic cells and antigen transport pathway in vivo mediated by an OVA@DDAB/PLGA nano-vaccine. Journal of Nanobiotechnology, 19, 394.

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CFSE-based T-cell Proliferation Assay

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Thawed PBMC were resuspended in PBS 1X and labeled with 0.6 μM CFSE (Molecular Probes, Eugene, Oregon). CFSE labeled PBMCs were stimulated with 2 μg/mL of HIV consensus B peptides identified in the ELISPOT assay; Gag7876 (EKIRLRPGGKKKYKL) for subjects NF-1042 and KBC-1035, Gag937 (IYKRWIILGLNKIVR) for subject RJP-1038 and Pol5683 (TAVQMAVFIHNFKRK) for subject ST-1041, in RPMI-1640 containing 10% human AB serum (Gemini, Burlington, ON). Stimulation with media alone served as a negative control, whereas stimulation with 25 ng/ml of Staphylococcol enterotoxin B (SEB) (Sigma-Aldrich) and 2 μg/mL of CEFT (CMV, EBV, Influenza and Tetanus peptides) were used as positive control stimulations. Monoclonal antibodies directed against immune checkpoint molecules (PD-1, CD160 or HVEM) along with their corresponding isotype controls were added to the culture conditions at 5 μg/mL. All stimulatory conditions were tested in quadruplicates. Following six days of incubation at 37°C and 5% CO₂, cells were monitored for viability with the Trypan blue exclusion test and further stained for cell surface markers using Live/Dead (Molecular Probes), αCD3, αCD8 (ebioscience), and αCD4 mAbs (BD Biosciences, Mississauga, ON). PBMCs were acquired using a BD LSRII flow cytometer and analyzed with FlowJo software version 9.4.11 (FlowJo LLC, Ashland, Oregon).

El-Far M., Pellerin C., Pilote L., Fortin J.F., Lessard I.A., Peretz Y., Wardrop E., Salois P., Bethell R.C., Cordingley M.G, & Kukolj G. (2014). CD160 isoforms and regulation of CD4 and CD8 T-cell responses. Journal of Translational Medicine, 12, 217.

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Multiparametric Flow Cytometry of Immune Cells

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Flow-cytometric analysis of single-cell suspensions from spleen, lymph nodes (inguinal, axillary, cervical and mesenteric lymph nodes), thymus, whole blood, bone marrow, and TILs was performed using α-CD45, α-CD8, α-CD4, α-CD19, α-CD44, α-CD25, α-CD122, α-FoxP3, α-IFN-γ (all eBioscience), α-TNF-α, α-Ki67, α-CD3 (BioLegend), α-NK1.1 (BD Pharmingen). Stainings for intracellular FoxP3, Ki67, TNF-α, and IFN-γ were performed following the manufacturer’s instructions (eBioscience).

Jing H., Hettich M., Gaedicke S., Firat E., Bartholomä M, & Niedermann G. (2019). Combination treatment with hypofractionated radiotherapy plus IL-2/anti-IL-2 complexes and its theranostic evaluation. Journal for Immunotherapy of Cancer, 7, 55.

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Multiparameter Flow Cytometry of Lymphoid Tissues

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Flow cytometric analysis of single-cell suspensions from lymph nodes, spleen, tumor-infiltrating lymphocytes (TILs), and tumor cells was performed using α-PD-1, α-PD-L1, α-CD45 (all BioLegend), α-CD3 (BD Pharmingen), α-CD4, α-CD8, α-CD19 (all eBioscience), α-PD-1-AF680, and α-PD-L1-AF680. Adipocytes were identified after BAT dissociation (as described above) and subsequent staining with α-PAT-2 (mouse IgM; Santa Cruz) and α-mouse IgM (eBioscience) as secondary antibodies. Cells were analyzed using a BD FACSVerse flow cytometer with FACSuite software (Becton Dickinson).

Hettich M., Braun F., Bartholomä M.D., Schirmbeck R, & Niedermann G. (2016). High-Resolution PET Imaging with Therapeutic Antibody-based PD-1/PD-L1 Checkpoint Tracers. Theranostics, 6(10), 1629-1640.

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Multiparametric Flow Cytometry Analysis

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Cells were labeled with fluorescence antibodies for mouse α-CD4, α-CD8, α-INF-γ, α-IL-2, α-IL-10, α-IL-4, α-IL-17A, α-CD44, and α-CD62L (eBioscience, CA). To assess immune responses after transplantation, total splenocytes or isolated CD4⁺ T cells were seeded on 48 well plates and stimulated in complete media for 4 hrs at 37°C with phorbol 12-myristate 13-acetate (PMA; 50 ng/ml; Sigma-Aldrich), Ionomycin (500ng/ml; Sigma-Aldrich) and Brefeldin A (eBioscience). Thereafter, cells were fixed, permeabilized and stained with respective antibodies at a concentration of 1–5 μg per 10⁶ cells. Flow cytometry measurements were performed on a FACS Canto II (BD Bioscience, CA, USA) and data were analyzed using FlowJo (FlowJo Software, OR, USA).

Krenzien F., Quante M., Heinbokel T., Seyda M., Minami K., Uehara H., Biefer H.R., Schuitenmaker J.M., Gabardi S., Splith K., Schmelzle M., Petrides A.K., Azuma H., Pratschke J., Li X.C., ElKhal A, & Tullius S.G. (2016). Age-dependent metabolic and immunosuppressive effects of Tacrolimus. American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons, 17(5), 1242-1254.

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Comprehensive Immune Cell Phenotyping

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The following antibodies were used: α-CD45 (BioLegend, Cat. #:103149), α-CD8 (Invitrogen, Cat. #:45–0081-80), α-CD4 (Invitrogen, Cat. #:25–0042-82), α-CD3 (BioLegend, Cat. #:100341), α-CD3 (BioLegend, Cat. #:100206), α-CD107a (BioLegend, Cat. #:328626), α-CD56 (BioLegend, Cat. #:318322), α-NKG2D (BioLegend, Cat. #:320820) and PE-conjugated goat anti-human IgG Fc secondary antibodies (Southern Biotech, Birmingham, USA).

Kuang Z., Li L., Zhang P., Chen B., Wu M., Ni H., Yi S., Zou J, & Liu J. (2020). A novel antibody targeting TIM-3 resulting in receptor internalization for cancer immunotherapy. Antibody Therapeutics, 3(4), 227-236.

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Multiparametric Immune Cell Analysis

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Splenocytes were labeled for surface and intracellular antigens with fluorescence α-CD4, α-CD8, α-CD25, α-FOXP3, α-IFN-γ, α-IL17 and α-IL10 anti-mouse antibodies (Invitrogen). Intracellular cytokine staining for FOXP3 was performed with a commercially available staining kit (Invitrogen). Flow cytometry measurements were performed on a FACS Canto II (BD Bioscience); data were analyzed using FlowJo (FlowJo Software, OR).

Maenosono R., Nian Y., Iske J., Liu Y., Minami K., Rommel T., Martin F., Abdi R., Azuma H., Rosner B.A., Zhou H., Milford E., Elkhal A, & Tullius S.G. (2021). Recipient Sex and Estradiol levels affect transplant outcomes in an age-specific fashion. American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons, 21(10), 3239-3255.

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