Anti thy1

Manufactured by BioLegend

Anti‐Thy1.2 is a monoclonal antibody that binds to the Thy1.2 antigen expressed on the surface of mouse cells. It is commonly used in flow cytometry and cell separation applications to identify and isolate specific cell populations.

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4 protocols using anti thy1

Comprehensive Immune Profiling of Tumor Samples

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Exactly 0.5 to 1 × 10⁶ tumour single‐cell suspension or the treated cells were incubated with an antibody cocktail. Following reagents and antibodies were used for flow cytometry analysis, including Zombie Red™ Fixable Viability Kit, anti‐CD45, anti‐F4/80, anti‐CD11b, anti‐CD206, anti‐CD80, anti‐Thy1.2, anti‐CD4, anti‐CD8a, anti‐PD‐1, anti‐Ly6G, anti‐Ly6C and anti‐SIRPα (BioLegend). For intracellular staining, the cells were first stained for membrane‐bound protein, then treated with Fixation/Permeabilisation buffer (BD Pharmingen San Jose, California) and incubated with anti‐TNF‐α, anti‐IL‐6 and anti‐IL‐17A. Flow cytometry was performed on BD FACSAria™ III instruments (BD Biosciences, Franklin Lakes, New Jersey). During running on flow cytometry instrument, 20,000 events were recorded for each sample. Fluorescence compensation was made by single positively stained control on BD compensation beads. Data were analysed by FlowJo software, version 10.4 (Tree Star Inc.). The stained samples were carefully gated according to FMO controls (Fluorescence minus one control). Gating strategies were described in Figure S1.

Pan L., Wang B., Chen M., Ma Y., Cui B., Chen Z., Song Y., Hu L, & Jiang Z. (2022). Lack of SIRP‐alpha reduces lung cancer growth in mice by promoting anti‐tumour ability of macrophages and neutrophils. Cell Proliferation, 56(2), e13361.

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Single-Cell Immune Phenotyping

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Single-cell suspensions were stained using anti-CD8a (Biolegend) and anti-Thy1.2 (Biolegend) followed by streptavidin-APC (eBioscience). Data were acquired on an LSR Fortessa or an Aria Sorter (Becton Dickinson).

Hutter K., Lohmüller M., Jukic A., Eichin F., Avci S., Labi V., Szabo T.G., Hoser S.M., Hüttenhofer A., Villunger A, & Herzog S. (2020). SAFB2 Enables the Processing of Suboptimal Stem-Loop Structures in Clustered Primary miRNA Transcripts. Molecular cell, 78(5).

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Multicolor Flow Cytometry for Immune Cell Analysis

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Enzymatically digested cell suspensions were incubated for 15 min at
4°C in PBS with 0.5% FCS with the following antibodies: anti-CD16/32,
anti-CD8α, anti-CD11b, anti-CD11c, anti-CD45.1, anti-CD45.2,
anti-CD103, anti-CD169, anti-CD301b, anti-Thy1.1 (all from Biolegend),
anti-PDL2 (BD Biosciences), or anti-SIGN-R1 (R&D Systems). Viability was
determined using Fixable Viability Dye eFluor 780 (Invitrogen). Experiments
examining CCR7 were done by first incubating cells with anti-CCR7 (BD
Biosciences) for 30 min at 37°C prior to additional surface staining
as above. Intracellular cytokine staining was done by first incubating cells
in PMA/Ionomycin (Sigma) in the presence of GolgiPlug for 4 hours prior to
surface staining followed by intracellular staining using BD
Cytofix/Cytoperm kit (BD Biosciences). hCCL1-AF647 (Almac) binding was
accomplished by first incubating 2×10⁶ cells in 50
μl of 0.25 μg/ml hCCL1-AF647 in PBS/0.5% FCS/20mM HEPES for 1
hr at 37°C. Cells then underwent additional antibody staining as
above. All samples were run on BD Fortessa X-20 or BD LSRII and analyzed
using FACS Diva 8 (BD Biosciences) and FlowJo (Version 10) (TreeStar).

Sokol C.L., Camire R.B., Jones M.C, & Luster A.D. (2018). The chemokine receptor CCR8 promotes the migration of dendritic cells into the lymph node parenchyma to initiate the allergic immune response.. Immunity, 49(3), 449-463.e6.

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Evaluating Transduction Efficiency in T Cells

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Staining steps to evaluate transduction efficiency were performed at room temperature for 12 minutes, with PBS washes between steps. Cells were costained with either eFluor 780 (eBioscience, 65–0865–15) or eFluor450 (eBioscience, 65–0863–18) fixable viability dye depending on the experimental condition. Transduced human T cells were detected by staining for the RQR8 marker, utilizing anti-CD34 (R&D system, FAB7227G, RRID:AB_10973657). Murine CAR T-cell transduction was evaluated by staining for THY1.1 marker, utilizing anti-Thy1.1 (BioLegend, 202522, RRID:AB_1595477). The samples were stained in 96-well plates and resuspended in 100 μL of PBS. Flow cytometry was performed using the MacsQuantX flow cytometer (Miltenyi Biotec), running 50 μL of cell suspension. Data analysis was conducted using FlowJo v10 (Treestar, RRID:SCR_008520).

Righi M., Gannon I., Robson M., Srivastava S., Kokalaki E., Grothier T., Nannini F., Allen C., Bai Y.V., Sillibourne J., Cordoba S., Thomas S, & Pule M. (2023). Enhancing CAR T-cell Therapy Using Fab-Based Constitutively Heterodimeric Cytokine Receptors. Cancer Immunology Research, 11(9), 1203-1221.

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