Tim3 bv421

Manufactured by BD

Sourced in United States

Tim3-BV421 is a fluorochrome-conjugated antibody used for flow cytometry analysis. It is designed to detect the Tim3 receptor, which is expressed on various immune cell types. The BV421 fluorochrome enables the visualization and quantification of Tim3-positive cells in a sample.

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5 protocols using tim3 bv421

Flow Cytometric Analysis of T-cell Markers

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Flow cytometry was performed on peripheral blood T cells with the following antibodies: PD1-BV510 (BD Biosciences, #563076), Tim3-BV421 (BD Biosciences, #565562), and LAG3-PerCP-eFluor 710 (BD Biosciences, #3DS223H). Samples were washed twice and flow cytometry was conducted with FACS Verse (BD Biosciences). Due to lack of available samples, only seven peripheral blood samples were analyzed from the osteosarcoma group and 8 peripheral blood samples were analyzed from the Ewing sarcoma group.

Si S.J., Wertheim G.B, & Barrett D.M. (2021). High Expression of TIM 3 and Galectin 9 on Immunohistochemistry Staining of Tumor Specimen at Diagnosis in Pediatric Patients with Ewing Sarcoma. Journal of cancer immunology, 3(3), 163-176.

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Characterizing Immune Cell Profiles in NSCLC Tumors

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For binding ability of CHI3L1 to receptor, NSCLC cell line H1299 was harvested and thoroughly mixed with His-tagged rCHI3L1 or nCHI3L1 Ab for 1 h. Subsequently, cells were incubated with FITC-labeled 6x-His Tag antibody at 4 °C for 1 h and analyzed by FACS. To determine PD-1 and TIM-3 presentation on CD8⁺ T cells, cells were stained using anti-human PD-1-BB515 (BD Bioscience) and TIM-3-BV421 (BD Bioscience) antibodies. After cell surface staining, intracellular CD107a was stained with CD107a-PE (BD Bioscience) antibodies to detect T cell activities.
Allograft tumor tissues were digested with 0.1 mg/mL collagenase (Sigma-Aldrich) and 1 mg/ml dispase II (Sigma-Aldrich) at 37 ℃ for 30 min and meshed. Resulting single-cell suspension were stained with CD4, CD8, CD11b, CD25, CD86, CD206, CTLA4 and Foxp3 antibody (BD Bioscience). The data were recorded by CytoFLEX (Beckman Coulter). The detailed antibodies conditions are listed in Table S2.

Yang P.S., Yu M.H., Hou Y.C., Chang C.P., Lin S.C., Kuo I.Y., Su P.C., Cheng H.C., Su W.C., Shan Y.S, & Wang Y.C. (2022). Targeting protumor factor chitinase-3-like-1 secreted by Rab37 vesicles for cancer immunotherapy. Theranostics, 12(1), 340-361.

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Immunophenotyping of T-cell subsets in PBMC

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Human peripheral blood samples were collected in 4 heparin tubes at baseline, following nivolumab infusion and multiple timepoints following TIL infusion. Peripheral blood mononuclear cells (PBMC) were collected using a Ficoll gradient and cryopreserved in 10% DMSO and FBS. Cells were thawed in media, and subsequently stained in PBS containing 5% FBS (vol/vol, FACS buffer) with: CD3 BUV496, CD56 BUV563, CD4 BUV737, CD197 BV421, CD28 BV480, CD14 BV605, CD19 BV605, CD95 BV711, CD195 BV786, CD127 PE, CD194 PE-Cy7, CD45RA Alexa488, CD25 PerCP-Cy5.5, NKG2D APC, Tim3 BV421, PD1 BV480, CD226 BV711, CTLA4 BV786, Lag3 PE, TIGIT PE-Cy7, CD244 Alexa488, CD27 PerCP-Cy5.5, BTLA APC from BD Biosciences. Dead cells were excluded using the Zombie NIR Fixable Viability Kit from Biolegend, incubated at 4 °C for 1 hr, then washed twice with FACS buffer, and finally fixed in PBS containing 1% paraformaldehyde before running flow cytometry. Cells were acquired on a BD FACSymphony™ A5, and data were analyzed with FlowJo Version 10.0 software. All cell gates were drawn uniformly for analysis across patients and time points, with gating strategy provided in Supplementary Appendix 2. Plots of t-SNE were generated by Flowjo Version 10.6.1 according to the expression of CD45RA, CCR7, CD28 and CD95. Different memory T cell subsets were shown using separate colors.

Creelan B.C., Wang C., Teer J.K., Toloza E.M., Yao J., Kim S., Landin A.M., Mullinax J.E., Saller J.J., Saltos A.N., Noyes D.R., Montoya L.B., Curry W., Pilon-Thomas S.A., Chiappori A.A., Tanvetyanon T., Kaye F.J., Thompson Z.J., Yoder S.J., Fang B., Koomen J.M., Sarnaik A.A., Chen D.T., Conejo-Garcia J.R., Haura E.B, & Antonia S.J. (2021). Tumor-infiltrating lymphocyte treatment for anti-PD-1 resistant metastatic lung cancer: a phase I trial. Nature medicine, 27(8), 1410-1418.

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Comprehensive Immune Profiling in SLE and RA

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PBMCs from patients with SLE or RA and HC were collected and analyzed immediately for the molecular phenotypes of lymphocytes. The antibodies used for the surface marker analysis include anti-human CD3-ECD (Beckman Coulter, USA), CD4-Percp-Cy5.5, CD8-PE-Cy7, PD1-FITC, TIGIT-APC, ICOS-PE, TIM3-BV421, CD19-V500, CD3-FITC, CD8-V500, CD25-APC, HLA-DR- PE-Cy7, CD69-V450, and CD127-PE (BD Biosciences, USA). Briefly, 50 μl of cells was incubated with appropriate antibodies on ice in the dark for 30 min, followed by washing in PBS. All the samples were analyzed with a Navios flow cytometer (Beckman Coulter) and Kaluza analysis software (Beckman Coulter).

Zhou H., Li B., Li J., Wu T., Jin X., Yuan R., Shi P., Zhou Y., Li L, & Yu F. (2019). Dysregulated T Cell Activation and Aberrant Cytokine Expression Profile in Systemic Lupus Erythematosus. Mediators of Inflammation, 2019, 8450947.

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Phenotypic Analysis of Activated PBMC Subsets

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The treated PBMCs were then washed twice in phosphate buffer saline (PBS) and stained with BD Multitest™ CD4 (clone SK3) FITC/CD38 (clone HB7) PE/CD3 (clone SK7) PerCP/Anti-HLA-DR (clone L243) APC (BD Biosciences, San Jose, CA), Tim-3 BV421 (Clone 7D3, BD Biosciences, San Jose CA), PD-1 PE-Cy7 (Clone EH12.1, BD Biosciences, San Jose CA), CD69 APC-H7 (Clone FN50, BD Biosciences, San Jose, CA) and LIVE/DEAD™ Fixable Aqua Stain (Invitrogen, Carlsbad, CA). The PBMCs were then fixed with 2% paraformaldehyde (PFA), washed again in PBS and events acquired using a BD FACS Canto II. Fluorescence minus one (FMO) controls were used to set cut-off gates for markers that did not have a clear resolution during data analysis (Supplementary Figure 1).

Olwenyi O.A., Asingura B., Naluyima P., Anywar G.U., Nalunga J., Nakabuye M., Semwogerere M., Bagaya B., Cham F., Tindikahwa A., Kiweewa F., Lichter E.Z., Podany A.T., Fletcher C.V., Byrareddy S.N, & Kibuuka H. (2021). In-vitro Immunomodulatory activity of Azadirachta indica A.Juss. Ethanol: water mixture against HIV associated chronic CD4+ T-cell activation/ exhaustion. BMC Complementary Medicine and Therapies, 21, 114.

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