Thy 1

Manufactured by Thermo Fisher Scientific

Sourced in United States

Thy-1.2 is a lab equipment product from Thermo Fisher Scientific. It is a membrane glycoprotein that serves as a cell surface marker. The core function of Thy-1.2 is to facilitate cell-cell and cell-matrix interactions.

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Lab products found in correlation

7 protocols using thy 1

Immunophenotypic Analysis of Murine Hematopoietic Cells

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Bone marrow (BM) and spleen cells were stained with either PE- or APC-conjugated monoclonal antibodies specific for Ter119, CD71, CD61, CD41, Mac-1, Gr-1, B220 or Thy-1 (purchased from eBioscience or Biolegend, San Diego, CA, USA) for 20 min on ice. Flow cytometry was performed with an LSRII (Beckton-Deckinson, San Diego, CA, USA) and analyzed by using FlowJo software (TreeStar, Ashland, OR, USA). For stem/progenitor analysis, BM or spleen cells were stained with antibodies against c-Kit, Sca-1, CD34, CD16/32 (FcγR II/III), and antibodies against lineage (Lin) markers including CD3, CD4, CD8α, CD19, B220, Gr-1, CD127 and Ter119 (from eBioscience or Biolegend, San Diego, CA, USA) for 1 hour on ice followed by flow cytometric analysis.

Akada H., Akada S., Hutchison R.E, & Mohi G. (2014). Loss of wild-type Jak2 allele enhances myeloid cell expansion and accelerates myelofibrosis in Jak2V617F knock-in mice. Leukemia, 28(8), 1627-1635.

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Live Imaging and Immunostaining of Cardiac Organoids

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For live imaging, single cardiac organoids were plated in round bottom ultra-low plates (Cat# 7007, Corning, Inc). Each well was imaged every hour for GFP and RFP expression up to 96 h using a BZ-9000 Fluorescence Microscope (Keyence). For EdU analysis, Click-it EdU kit (Life Technologies) was used followed by immunostaining with primary and secondary antibodies. For whole-mount staining, embryos were fixed in 4% paraformaldehyde overnight and then 30% sucrose and then incubated with primary and secondary antibodies. For immunohistochemistry, embryos were fixed in 4% paraformaldehyde overnight and then 30% sucrose, and then embedded in OCT, sectioned and stained using standard protocols. Antibodies used were: mouse α-Islet1 (1:200; Cat. 39.3F7 Developmental Studies Hybridoma Bank, Iowa City, IA), rat α-RFP (1:200; Cat. 5F8 Chromotek), chicken GFP (1:500; Cat A10262 Invitrogen), rabbit Cxcr4 (1:500; Cat. 119-15995 Biotrend), rabbit αSMA (1:200; Cat. Ab5694 Abcam), Pecam-1 (1:100; Cat. 553371 BD Biosciences), Thy1 (Cat. 17-0902-82 eBiosciences), mouse cardiac TnT (1:500; Cat. MS-295-P1Thermo Fisher). Alexa Fluor secondary antibodies (1:500; Life Technologies) were used for secondary detection and images were acquired with an Evos fl microscope.

Andersen P., Tampakakis E., Jimenez D.V., Kannan S., Miyamoto M., Shin H.K., Saberi A., Murphy S., Sulistio E., Chelko S.P, & Kwon C. (2018). Precardiac organoids form two heart fields via Bmp/Wnt signaling. Nature Communications, 9, 3140.

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Comprehensive Immune Cell Phenotyping

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Antibodies used include: Ii(In-1, 1:100), H2-M (2E5A, 1:50), Rat IgG1 (R3–34, 1:50) (all from BD Biosciences); podoplanin (8.1.1, Biolegend, 1:500); CLIP (15G4, Santa Cruz, 1:5); H2-O^{69 (link)} (Mags.Ob3, Lisa Denzin, 1:400); 10.1.1 (UVA lymphocyte culture center, 1:1000); CD45 (30-F11, 1:1000), CD31 (390, 1:1000), MHC-II (M5/114.15.2, 1:50 (Fig 1) to 1:500 (Fig 6)), CD8 (53-6.7, 1:1000), CD4 (GK1.5, 1:1000), Thy1.1 (HIS51, 1:1000), Thy1.2 (53-2.1, 1:1000), CD45.1 (A20, 1:500), CD11c (N418, 1:500), CD11b (M1/70, 1:500), CD69 (H1.2F3, 1:500), CD62L (MEL-14, 1:1000), CD44 (IM7, 1:1000), CD25 (PC61.5, 1:500), Y-AE (eBioY-Ae, 1:200), PD-1 (RMP1–30, 1:100), LAG-3 (eBioC9B7W, 1:100), Rat IgG2b (eB149/10H5) (all from eBioscience). Intracellular staining for PD-1, LAG-3, Ii, H2-M and H2-O was done using the Cytofix/Cytoperm kit (BD Bioscience), and Ki67 (SolA15, 1:500) and FoxP3 (FJK-16s, 1:100) were stained using Treg permeabilization buffers (eBioscience). Annexin V (1:20) was stained using the eBioscience kit. DAPI (Sigma) or live/dead aqua (Invitrogen, 1:200) were used to distinguish live cells. Cells were acquired on a FACSCanto II (BD Biosciences) and data analyzed using FlowJo (Tree Star).

Rouhani S.J., Eccles J.D., Riccardi P., Peske J.D., Tewalt E.F., Cohen J.N., Liblau R., Mäkinen T, & Engelhard V.H. (2015). Roles of lymphatic endothelial cells expressing peripheral tissue antigens in CD4 T-cell tolerance induction. Nature Communications, 6, 6771.

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Stromal Cell Immunophenotyping by FACS

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Single cell suspensions of stromal cells were incubated with anti-CD16/CD31 blocking mAb (2.4G2, Bio Xcell) and then with antibodies against gp38 (BioLegend) and CD31 (eBioscience). Additional antibodies included those specific for CD45, PD-L1 (both from BioLegend), Lyve-1, ICAM-1, MAdCAM-1, and LtβR (all from eBioscience). LNSC were stained with Dapi to discriminate live and dead cells. For experiments involving the co-culture or transfer of FH T cells, Thy1.2 (eBioscience), CD45.1 (eBioscience), or Tyr₃₆₉-tetramer were used as identifying markers. Flow cytometry was performed on a FACSCanto II (BD Biosciences) and data analyzed using FloJo software (Tree Star).

Cohen J.N., Tewalt E.F., Rouhani S.J., Buonomo E.L., Bruce A.N., Xu X., Bekiranov S., Fu Y.X, & Engelhard V.H. (2014). Tolerogenic Properties of Lymphatic Endothelial Cells Are Controlled by the Lymph Node Microenvironment. PLoS ONE, 9(2), e87740.

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Isolation and Analysis of Innate Lymphoid Cells

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The SI of P1 mice were microdissected, minced, and digested with 2 mg ml⁻¹ collagenase D (Roche). Cell suspensions were passed through a 70-μm cell strainer and mononuclear cells were isolated. Cells were pre-incubated with anti-mouse CD16/CD32 for blockade of Fc receptors, and incubated with the appropriate monoclonal Ab conjugates. Propidium iodide (Sigma-Aldrich) or DAPI (4′,6-diamidino-2-phenylindole) (Invitrogen) was used to distinguish live cells from dead cells during cell analysis. Stained cells were analyzed on a FACS Canto or LSRII machine using the Diva software (BD Bioscience). Data were analyzed with FlowJo software (TreeStar). ILC3 cells were defined as CD45⁺Lin⁻Thy1⁺Sca-1^hi as described^{11 (link)}. The following fluorochrome-conjugated anti-mouse antibodies were used at indicated dilutions: Thy-1.2 (53-2.1, 1:200), Sca-1 (D7, 1:200), CD45 (30-F11, 1:200), CD11b (M1/70, 1:200), IL-22 (1H8PWSR, 1:100), IL-17A (eBio17B7, 1:100) were from eBioscience; lineage cocktail (CD3, B220, CD11b, Gr-1, Ter119⁻) (145-2C11, RA3-6B2, M1/70, RB6-8C5, TER-119, 1:100) and CX3CR1 (SA011F11, 1:200) were from BioLegend.

Chen L., Strohmeier V., He Z., Deshpande M., Catalan-Dibene J., Durum S.K., Moran T.M., Kraus T., Xiong H., Faith J.J., Sodhi C.P., Hackam D.J., Lira S.A, & Furtado G.C. (2019). Interleukin 22 disrupts pancreatic function in newborn mice expressing IL-23. Nature Communications, 10, 4517.

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Multiparametric Flow Cytometry Analysis

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Fluorescent antibodies were purchased from Biolegend (CD3, γδTCR, CD4, CD8, CD27, Vγ4, Vγ5, IL-17A, IFN-γ, CD25, CD73, Thy-1.2, Anti-rat IgM), eBioscience (Fixable Viability Dye, RORγt, c-Maf, CD44, CD24), and Cell Signaling Technologies (p-Drp1). For cell surface marker staining, cells were first treated with Fc Blocker and then stained with cell surface antibodies at 4°C for 20 min. The relevant isotype control antibodies were also used. For intracellular cytokine staining, cells were stained with surface antibodies followed by fixation, permeabilization (Biolegend), and finally intracellular staining of IL-17/IFN-γ at 4°C for 2h. For c-Maf/RORγt staining, cells were stained with surface antibodies (Abs), and then fixed and permeabilized with Foxp3/Transcription Factor Staining kit (eBioscience) for 45 min followed by intranuclear staining of c-Maf/RORγt at 4°C for 2h.

Wang Y., Qin H., Cai Y., Chen X., Li H., Montoya-Durango D.E., Ding C., Hu X., Chariker J.H., Sarojini H., Chien S., Rouchka E.C., Zhang H.G., Zheng J., Qiu F, & Yan J. (2023). Natural γδT17 cell development and functional acquisition is governed by the mTORC2-c-Maf-controlled mitochondrial fission pathway. iScience, 26(5), 106630.

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Isolation and Characterization of Murine Mesenchymal Stem Cells

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BM cells obtained as described above were stained with monoclonal anti-mouse antibodies for CD3, CD11b, CD11c, CD19, and CD34 for negative selection and for CD73 (ecto-5′-nucleotidase), CD90.2 (Thy-1.2) and CD105 (endoglin; eBioscience, San Diego, CA, USA) for positive selection. The following subpopulations were sorted using a BD FACSAria™ II and used for transplantation assays: (a) CD73⁺; (b) CD90.2⁺; and (c) CD105⁺.

Silveira G.D., Ishimura M.E., Teixeira D., Galindo L.T., Sardinha A.A., Porcionatto M, & Longo-Maugéri I.M. (2019). Improvement of Mesenchymal Stem Cell Immunomodulatory Properties by Heat-Killed Propionibacterium acnes via TLR2. Frontiers in Molecular Neuroscience, 11, 489.

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