Anti klrg 1

Manufactured by Thermo Fisher Scientific

Anti-KLRG-1 is a laboratory reagent used for the detection and analysis of KLRG-1 (Killer cell lectin-like receptor G1) expression in cells. KLRG-1 is a cell surface receptor that plays a role in the regulation of immune responses. This reagent can be utilized in various research applications involving the study of KLRG-1 and its associated cellular functions.

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4 protocols using anti klrg 1

Characterization of Antigen-Specific T Cell Responses

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Flow cytometry and tetramer were performed as previously described^{55 (link)}. Briefly, single cell suspended splenocytes were incubated with gp33-tetramer and np396-tetramer for 15 minutes and gp-66 tetramer for 30 minutes at 37 °C. After the incubation, surface antibodies were added, including anti-CD8 or anti-CD4 along with anti-IL7R, anti-PD-1, anti-CD62L, anti-CD44, anti-KLRG-1, anti-TIM3, anti-Lag-3, and anti-2B4 antibodies (eBioscience) for 30 minutes at 4 °C. For intracellular cytokine stain single suspended splenocytes were incubated with the LCMV specific peptides gp33 and np396 or the MHC-II epitope gp61. After 1 h, Brefeldin A (eBioscience) was added, followed by an additional 5 h incubation at 37 °C. After surface stain with anti-CD8 or anti-CD4 antibodies (eBioscience) cells were fixed with 2% formalin and permeabilized with 0.1% Saponin and stained with anti-IFNγ and anti-TNF antibodies (eBioscience) for 30 min at 4 °C.

Lang E., Pozdeev V.I., Shinde P.V., Xu H.C., Sundaram B., Zhuang Y., Poschmann G., Huang J., Stühler K., Pandyra A.A., Keitel V., Häussinger D., Lang K.S, & Lang P.A. (2018). Cholestasis induced liver pathology results in dysfunctional immune responses after arenavirus infection. Scientific Reports, 8, 12179.

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Lung and Tumor Immune Cell Isolation

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Lymphocytes were isolated from lung and tumor tissue by digestion with collagenase A (1 mg/ml; Roche) and DNase I (0.5 µg/ml; Roche) in isolation buffer (RPMI 1640 supplemented with 5% FBS, 1% l-glutamine, 1% penicillin-streptomycin, and 10 mM Hepes) for 30 min at 37°C. Cells were filtered through 100-µm cell strainers, washed in isolation buffer, and stained in PBS supplemented with 0.25% BSA, 2 mM EDTA, and 0.1% sodium azide. Antibodies used included anti-CD45, anti-Foxp3, anti–IL-18Rα, anti-ST2, anti-CD62L, anti-CD103, anti–PD-1, anti-GITR, anti–CTLA-4, anti-KLRG1, anti-Ki67, anti-CD5, anti-NK1.1, anti-CD45R (B220), anti–IL-17, anti–IL-4, anti-IFNγ, and anti–Ly-6C (eBioscience); anti-TCRβ, anti-CD3, anti-CD4, anti-CD8, anti-CD127, anti-CD11B, anti-MHCII, and anti-Gr1 (BioLegend); anti-CD44 and anti-CD25 (Tonbo); anti–IL-5 and anti-TNFα (BD PharMingen); and anti-AREG (R&D Systems). For exclusion of dead cells, samples were first stained with Ghost Dye (Tonbo) cell viability reagent. Intracellular staining for cytokines, Foxp3, AREG, Ki-67, and CTLA-4 was performed using the Foxp3/transcription factor staining buffer set (eBioscience) as per manufacturer’s protocol. The H2-K^b OVA_257–264 tetramer was obtained from the NIH Tetramer Core Facility.

Green J.A., Arpaia N., Schizas M., Dobrin A, & Rudensky A.Y. (2017). A nonimmune function of T cells in promoting lung tumor progression. The Journal of Experimental Medicine, 214(12), 3565-3575.

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Isolation and Characterization of Lung ILC2s and Th2 Cells

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The left lobe of lung tissue was digested with 50 μg/ml Liberase TM (Roche) and 1 μg/ml DNase I (Sigma) for 45 min at 37°C. After obtaining a cell suspension, lineage-negative cells were stained with lineage cocktail antibodies and anti-CD45 (eBioscience), anti-Sca-1 (eBioscience) and anti-KLRG1 (eBioscience) antibodies for 30 min. ILC2s were defined as CD45⁺Lineage⁻Sca-1⁺KLRG1⁺ cells (Moro et al., 2015 (link)). After staining the surface markers, cells were fixed, permeabilized and stained for intracellular IL-5 and IL-13 by their respective antibodies. For staining Th2 cells, the following antibodies were used in cell suspension: anti-CD45 (eBioscience), anti-CD4 (eBioscience) and anti-IL-4 (eBioscience) antibodies (Wang et al., 2014 (link)). IL-5⁺Th2 cells and IL-13⁺Th2 cells can be detected by added anti-IL-5 and IL-13 antibodies.

Xue L., Li C., Ge G., Zhang S., Tian L., Wang Y., Zhang H., Ma Z, & Lu Z. (2021). Jia-Wei-Yu-Ping-Feng-San Attenuates Group 2 Innate Lymphoid Cell-Mediated Airway Inflammation in Allergic Asthma. Frontiers in Pharmacology, 12, 703724.

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Phenotypic Characterization of Lymphocytes

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Cells isolated from spleens and graft-draining axillary and brachial lymph nodes (dLN) were stained with anti-CD4 (BD Biosciences), anti-CD8 (Invitrogen) and anti-Thy1.1 (BD Biosciences). For phenotypic analysis cells were also surface-stained with anti-PD-1 (BioLegend), anti-2B4 (BD Biosciences or eBioSciences), anti-Thy1.1 (BD Biosciences), anti-LAG-3 (BioLegend), anti-CD127 (BioLegend), anti-KLRG-1 (eBioSciences), anti-CD44 (BioLegend or BD Biosciences), and anti-CD48 (BioLegend). Absolute numbers of lymphocytes from the spleen and draining lymph nodes were calculated using a Cellometer Auto T4 Cell Viability Counter (Nexcelom) according to the manufacturer’s instructions. Samples were analyzed on an LSRII flow cytometer (BD Biosciences). Data was analyzed using FlowJo 9 software (Treestar, San Carlos, CA) and Prism 6 software (GraphPad Software Inc.). For intracellular cytokine staining, lymphocytes were restimulated ex vivo with 1 μg/mL phorbol 12-myristate 13-acetate (PMA) (Sigma Life Sciences) and 1 μg/mL ionomycin (Sigma Life Sciences) where indicated, in the presence of 1 μg/mL Brefeldin A (BD Biosciences) for 4 hours. The Fix/Perm intracellular staining kit (BD Pharmingen) was used to detect IL-2 (BD Biosciences), TNF (BioLegend), and IFN-γ (BD Biosciences), according to manufacturer’s instructions.

Laurie S.J., Liu D., Wagener M.E., Stark P.E., Terhorst C, & Ford M.L. (2018). 2B4 Mediates Inhibition of CD8+ T Cell Responses via Attenuation of Glycolysis and Cell Division. Journal of immunology (Baltimore, Md. : 1950), 201(5), 1536-1548.

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