Cd90.2 30 h12

Manufactured by BioLegend

CD90.2 (30-H12) is a mouse monoclonal antibody that recognizes the mouse Thy-1 (CD90.2) antigen. Thy-1 is a glycosylphosphatidylinositol (GPI)-anchored protein expressed on the surface of various cell types, including T cells, neurons, and hematopoietic stem cells. The CD90.2 (30-H12) antibody can be used to identify and isolate Thy-1-positive cells.

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CD90.2 (clone 30-H12, (4 citations)

4 protocols using cd90.2 30 h12

Colonic Immune Cell Profiling Protocol

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For analysis of colonic tissue, after measuring the colon length, the distal colon, 2 cm above the rectum, was cut longitudinally and washed with PBS. The tissue was minced and digested with 2 mg/mL collagenase (FUJI-FILM Wako Pure Chemical, Osaka, Japan) solution containing 10 μM DNase I (Sigma-Aldrich, St. Louis, MO, USA) in Dulbecco’s modified Eagle medium (DMEM)–GlutaMAX (Thermo Fisher Scientific), with shaking for 1 h at 37 °C. The digested tissue was filtered through a 70 μm cell strainer (CORNING, Corning, NY, USA), pelleted by centrifugation at 1800× g for 5 min at 4 °C, and resuspended in HBSS containing 2% FBS, 10 nM HEPES, and 1% penicillin/streptomycin (Thermo Fisher Scientific). The cell suspensions were stained with an antibody cocktail for 30 min on ice. The following antibodies were used: CD45 (30-F11), from BD Biosciences, and CD11b (M1/70) and F4/80 (BM8), from BioLegend (San Diego, CA, USA). The following antibodies were used for the analysis of MSC markers: CD73 (TY11.8), CD44 (1M7), Sca-1 (E13-161.7), and CD140a (APA5), from BD Biosciences, and CD90.2 (30-H12), from BioLegend. Data acquisition was performed using FACS AriaII (BD Biosciences), and all analyses were carried out using FlowJo software, version 10.6.1 (TreeStar).

Hisamatsu D., Itakura N., Mabuchi Y., Ozaki R., Suto E.G., Naraoka Y., Ikeda A., Ito L, & Akazawa C. (2023). CD73-Positive Cell Spheroid Transplantation Attenuates Colonic Atrophy. Pharmaceutics, 15(3), 845.

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Multiparametric Flow Cytometry of Liver NPCs

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1 x 10⁶ liver NPC cells were incubated with 100 μl of various antibodies diluted at optimal concentrations for 40 min at 4 °C. The following fluorochrome-conjugated antibodies were used: CD45.2 (104; Biolegend, 109831), CD45.1 (A20; Biolegend, 110741), CD45 (30-F11; Biolegend, 103126) F4/80 (BM8; Biolegend, 123114), CD11b (M1/70; Biolegend, 101227), CD9 (MZ3; Biolegend, 124805), GPNMB (CSTREVL; Thermo Fisher, 50-5708-82), CD90.2 (30-H12; Biolegend, 105335), CD4 (GK1.5; Biolegend, 100407), CD8 (53-6.7; Biolegend, 100734), PD-1 (29F.1A12; Biolegend, 135218), IL-2 (JES6-5H4; Biolegend, 503807). Liver macrophages were gated as CD45+F4/80^hiCD11b^int and CD45+F4/80^intCD11b^hi for KC or MDM, respectively. For intracellular staining, 1 x 10⁶ NPCs were cultured in complete RMPI 1640 medium with Brefeldin A and PMA/ionomycin for 6 hours before harvested for flow staining. After surface staining, fixed cells were permeabilized using Invitrogen transcription factor staining buffer set according to manufacturer’s protocol. Samples were analyzed using BD LSR cell analyzer at the Vision Research Core Facility at the University of Michigan Medical School or Attune NXT4 Flow Cytometer at MCDB research core facility at the University of Michigan. Data were analyzed using the CellQuest software (BD Biosciences) or Attene NXT software and Flowjo (Flowjo.com).

Zhang P., Chen Z., Kuang H., Liu T., Zhu J., Zhou L., Wang Q., Xiong X., Meng Z., Qiu X., Jacks R., Liu L., Li S., Lumeng C.N., Li Q., Zhou X, & Lin J.D. (2022). Neuregulin 4 suppresses NASH-HCC development by restraining tumor-prone liver microenvironment. Cell metabolism, 34(9), 1359-1376.e7.

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Tetramer-Based Enrichment of Antigen-Specific Cells

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PE- and APC-labeled peptide: I-A^b tetramers were obtained from the NIH Tetramer Core Facility. Peptides used were pMOG 38–49 (GWYRSPFSRVVH) and pApoB 978–993 (TGAYSNASSTESASY). Tetramer enrichment was performed following a previously described protocol.^{76 (link)} Briefly, thymocytes were incubated with PE- and APC-labeled tetramers (10 nM each) for 1 h at room temperature, and tetramer-bound cells were magnetically enriched using EasySep PE- and APC-positive selection kit II (StemCell Technologies). Cells were stained with antibodies against CD90.2 (30-H12, BioLegend), CD11b (M1/70, eBioscience), CD11c (N418, eBioscience), NK1.1(PK136, eBioscience), B220 (RA-3-6B2, eBioscience), TCRβ, CD8, and CD4, and analyzed on a flow cytometer.

Ushio A., Matsuda-Lennikov M., Kalle-Youngoue F., Shimizu A., Abdelmaksoud A., Kelly M.C., Ishimaru N, & Takahama Y. (2024). Functionally diverse thymic medullary epithelial cells interplay to direct central tolerance. Cell reports, 43(4), 114072.

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Comprehensive Immune Cell Phenotyping

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Antibodies against CD4 (RM4-5 or GK1.5), CD8 (53-6.7), CD45R/B220 (RA3-6B2), CD45.2 (104), TER-119 (TER-119), CD31 (390), gp38 (Podoplanin; 8.1.1), CD3 (17A2), Klrg1 (2F1), CD127 (A7R34), CD11c (N418), CD11b (M1/70), CD19 (6D5), NK1.1 (PK136), CD90.2 (30-H12), GR-1 (RB6-8C5), IFN-γ (XMG1.2), TNF (MP6-XT22) and IL-2 (JES6-5H4) were from Biolegend, Pharmingen and eBioscience. All fluorescently labelled monoclonal antibodies were used at a 1:100 dilution, except for gp38 (Podoplanin) and TER-119, which were diluted 1:1,000, and 1:10, respectively. Dead cells were excluded with Zombie UV Fixable Viability Kit (Biolegend, cat. no. 423108). OVA- (SIINFEKL) and P1A epitope- (LPYLGWLVF) specific CTLs were identified as peptide-MHC class I tetramers (TCMetrix) binding cells amongst CD8⁺B220⁻ lymphocytes. BD Trucount Absolute Counting Tubes were used to determine absolute cell counts in blood. Spleen lymphocyte counts
were determined in a Neubauer chamber. Peptide-MHC tetramer-binding CTLs were back calculated. Cytokine profiles after restimulation with peptide (SIINFEKL for OVA, TYLPANASL for Her2; ProImmune) were determined in intracellular cytokine assays11 (link). Samples were measured on BD LSRFortessa and Beckman Coulter Gallios flow cytometers and were analysed using FlowJo software (Tree Star, Ashland, OR).

Kallert S.M., Darbre S., Bonilla W.V., Kreutzfeldt M., Page N., Müller P., Kreuzaler M., Lu M., Favre S., Kreppel F., Löhning M., Luther S.A., Zippelius A., Merkler D, & Pinschewer D.D. (2017). Replicating viral vector platform exploits alarmin signals for potent CD8+ T cell-mediated tumour immunotherapy. Nature Communications, 8, 15327.

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