Tim3 apc

Manufactured by Thermo Fisher Scientific

Sourced in United States

TIM3-APC is a fluorophore-conjugated antibody that binds to the TIM3 (T-cell Immunoglobulin and Mucin Domain-containing Protein 3) receptor. TIM3 is an immune checkpoint protein expressed on the surface of various immune cells, including T cells, natural killer cells, and dendritic cells. The TIM3-APC antibody can be used for the detection and analysis of TIM3-expressing cells in flow cytometry applications.

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Lab products found in correlation

Cd4 buv395, by BD (4 mentions) 41bb percpef710, by Thermo Fisher Scientific (3 mentions) Live dead ghost dye 780, by Cytek Biosciences (3 mentions) Lag3 bv711, by BD (3 mentions) Pd1 pecf594, by BD (3 mentions) Cd8 buv805, by BD (3 mentions) Cd3 fitc, by BD (3 mentions) Ctla 4 pe, by Thermo Fisher Scientific (2 mentions) Immunomagnetic negative selection, by STEMCELL (1 mentions) Rainbow beads, by Spherotech (1 mentions) Cd8 buv395, by BD (1 mentions) Pd l2 apc, by Thermo Fisher Scientific (1 mentions) Cd45 v450, by BD (1 mentions) Cd19 buv737, by BD (1 mentions) Cd34 bv786, by BD (1 mentions) Cd38 pe, by Exbio (1 mentions) Hla dr fitc, by Thermo Fisher Scientific (1 mentions) Lightning link fluorescein conjugation kit, by Abcam (1 mentions) Histopaque 1077, by Merck Group (1 mentions) Brilliant stain buffer, by BD (1 mentions)

6 protocols using tim3 apc

Adoptive Transfer of OT-1 T Cells

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For adoptive transfer of OT-1 T cells into B6 mice, OT-1 splenocytes were harvested as described above. CD8 T cells were isolated using immunomagnetic negative selection (StemCell, Vancouver, Canada; 19853), rinsed and suspended in PBS, and 2 × 10⁶ cells were adoptively transferred into 6–10 wk old, female, B6 mice via intraperitoneal injection. The day following transfer, mice were immunized subcutaneously with an individual SIINFEKL APL (100 µg) in complete Freund’s adjuvant (Sigma, F5881) or vehicle. Mice were euthanized at the times indicated, spleens were collected, processed as described above, and analyzed via flow cytometry using the following antibodies: SIINFEKL H2K^b tetramer-BV421 (NIH Tetramer Core Facility), CD3-FITC (BD 555274), CD4-BUV395 (BD 563790), CD8-BUV805 (BD 564920), LAG3-BV711 (BD 563179), PD1-PECF594 (BD 562523), TIM3-APC (eBioscience 17-5871-82), 41BB-PerCPeF710 (eBioscience 46-1371-82), CTLA4-PE (eBioscience 12-1529-42), CD44-BV786 (BD 563736) and Live/Dead Ghost dye 780 (Tonbo 13-0865-T100) or corresponding fluorescently labeled IgG controls.
Data collected on different days was normalized using rainbow beads (Spherotech, Lake Forest, IL; RFP-30-5A).

Zahm C.D., Colluru V, & McNeel D.G. (2017). Vaccination with High-Affinity Epitopes Impairs Antitumor Efficacy by Increasing PD-1 Expression on CD8+ T Cells. Cancer immunology research, 5(8), 630-641.

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Phenotyping Leukemic Cells from AML Patients

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Primary cells from peripheral blood of AML patients with hyperleukocytosis were obtained by leukapheresis before therapy initiation. The leukapheretic products were diluted 10-fold in phosphate buffered saline (PBS), and the mononuclear cell fraction was separated using Histopaque-1077 (Sigma, #H8889). The cells were resuspended in RPMI 1640 medium with 10% fetal calf serum and with antibiotics (100 U/ml penicillin, 100 µg/ml streptomycin), and aliquots were used for analysis of surface markers by flow cytometry and for mRNA isolation.
The antibodies used were as follows: CD45-V450 (#560367), CD4-BUV395 (#564724), CD8-BUV395 (#563795), CD19-BUV737 (#564303), CD34-BV786 (#743534), and CD371-BB515 (#565926) from BD Biosciences; HLA-DR-FITC (#11-9952-42), TIM-3-APC (#17-3109-42), and PD-L2-APC (#17-5888-42) from eBioscience; CLIP-PE (sc-12725 PE) from Santa Cruz; PD-L1-PE (#1P-177-T100), CD47-FITC (#1F-225-T100), and CD38-PE (#1P-366-T100) from Exbio (Prague, Czech Republic). HLA class I antibody (Abcam, ab2217) was conjugated in house using the Lightning-Link Fluorescein Conjugation kit (#707-0010, Innova Biosciences).

Kuželová K., Brodská B., Marková J., Petráčková M., Schetelig J., Ransdorfová Š., Gašová Z, & Šálek C. (2022). NPM1 and DNMT3A mutations are associated with distinct blast immunophenotype in acute myeloid leukemia. Oncoimmunology, 11(1), 2073050.

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Splenocyte Activation and Phenotyping

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Splenocytes were cultured at 2 × 10⁶/mL in RPMI 1640 + l-glutamine, 10% FCS, penicillin/streptomycin (200 U/mL), 1% NaPyr, 1% HEPES, 50 µM β-MeOH, and the designated peptide (2 µg/mL). At the time points indicated, cells were stained with the following antibodies: CD3-FITC (BD 555274), CD4-BUV395 (BD 563790), CD8-BUV805 (BD 564920), LAG3-BV711 (BD 563179), PD1-PECF594 (BD 562523), TIM3-APC (eBioscience 17-5871-82), 41BB-PerCPeF710 (eBioscience 46-1371-82), CTLA4-PE (eBioscience 12-1529-42), Live/Dead Ghost dye 780 (Tonbo 13-0865-T100), or corresponding fluorescently labeled IgG controls. Cells were then fixed for 15 min at 4°C in cytofix (BD Biosciences, San Jose, CA; 554655), and frozen in FCS + 10% DMSO. After all time points were collected, cells from all times were thawed, rinsed and resuspended in PBS + 3% FCS + 1 mM EDTA and analyzed by flow cytometry. All antibodies used were at 1:100 dilutions and stained for 30 min at 4°C in a 1:4 dilution of brilliant stain buffer (BD 563794) in PBS + 3% FCS + 1 mM EDTA.

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CD8+ T Cell Activation Assay

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B cells or dendritic cells (DCs) were enriched from splenocytes of OT-1 or B6 mice inoculated with Flt3 ligand-expressing B16 tumor cells^{25 (link)} using PE-labeled antibodies specific for either CD19 or CD11c (StemCell, Seattle, WA, Cat.# 17,684) as previously described.^{26 (link)} Similarly, CD8 + T cells were isolated using a negative selection CD8 + T-cell isolation kit (StemCell, Cat. # 19,853). After enrichment, each APC subset, and a subset of purified T cells, were cultured as described above with 2 µg/mL SIINFELK or the HLA-A2 sequence from SSX2 (RLQGISPKI) as a nonspecific control peptide. Naïve OT-1 T cells were added to each cell type at a 1:1 ratio and incubated for three days, after which cells were stained and analyzed by flow cytometry with the following panel: CD3-FITC (BD 555,274), CD4-BUV395 (BD 563,790), CD8-BUV805 (BD 564,920), LAG-3-BV711 (BD 563,179), PD1-PECF594 (BD 562,523), TIM3–APC (eBioscience 17-5871-82), CTLA4–PECy7 (Tonbo 60-1522-U100), 41BB–PerCPeF710 (eBioscience 46–1371–82), and Live/Dead Ghost dye 780 (Tonbo, San Diego, CA 13–0865–T100).

Zahm C.D., Moseman J.E., Delmastro L.E, & G. Mcneel D. (2021). PD-1 and LAG-3 blockade improve anti-tumor vaccine efficacy. Oncoimmunology, 10(1), 1912892.

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Phenotypic Characterization of TILs

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Both Young TILs and rapid expanded TILs (REP-TILs) were stained using fluorochrome-labelled monoclonal antibodies (mAb; from BD Bioscience, unless indicated otherwise) CD3-BV510, CD4-PerCP, CD8-BV421, CD45RA-FITC, CD45RO-PE, CCR7-PE-Cy7, CD62L-APC, CD69-PE-Cy7, CD28-APC, CD16-FITC (Dako, Glostrup, Denmark), CD137-PE, CD56-PE-Cy7, Gamma-Delta-TCR-PE (Biolegend, California, United States), LAG-3-FITC (LS Bioscience, Seattle, WA, United States), BTLA-PE, PD-1-PE-Cy7, TIM-3-APC (eBioscience, California, United States) and analysed with a FACS Canto II instrument (BD Biosciences). The phenotypic subpopulations was investigated for statistical difference using GraphPad Prism, Wilcoxon matched-pairs signed rank test.

Westergaard M.C., Andersen R., Chong C., Kjeldsen J.W., Pedersen M., Friese C., Hasselager T., Lajer H., Coukos G., Bassani-Sternberg M., Donia M, & Svane I.M. (2019). Tumour-reactive T cell subsets in the microenvironment of ovarian cancer. British Journal of Cancer, 120(4), 424-434.

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Multiparametric Flow Cytometry of Tregs

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For analysis of in vivo experiments, cells were stained with antibodies against human CD45 APC (Invitrogen), CD4 ECD (Beckman Coulter), CD3 Pacific Blue (eBioscience) CD8 PE and CD25 PE-Cy7 (BD) and the viability dye 7-AAD (eBioscience). To analyse expression of Treg-associated markers, freshly sorted or expanded Tregs were stimulated for 15h with anti-CD3/anti-CD28 beads at a ratio of 1 bead to 5 cells, or left untreated. Cells were stained with 7-AAD and antibodies against GITR FITC (R&D Systems), CTLA-4 PE, CD69 APC-Cy7, CD25 PE-Cy7 (BD), TIGIT PE, OX-40 FITC, TIM-3 APC, CD39 PE, FOXP3 eFluor 450, Perforin APC (eBioscience) Helios AlexaFluor 647 (Biolegend), and CD4 ECD (Beckman Coulter). For intracellular antigens (FOXP3, Helios, Perforin, CTLA-4), cells were fixed and permeabilized using a Foxp3 Staining Buffer Set (eBioscience). Samples were acquired using a BD FACSCanto (BD Biosciences) and analyzed using FACSDiva software (BD Biosciences). For staining for BCL-XL and MCL1, Abcam anti-BCL-XL FITC (7B2.5; ab26148) and anti-MCL-1 Alexa Fluor 488 (Y37, ab197529) antibodies were used, respectively, following manufacturer's instructions. Briefly, the cells were fixed with 4% paraformaldehyde and permeablized with PBS/0.1% Tween. The cells were then blocked with 10% normal goat serum/0.3 M glycine, followed by incubation with the antibody.

Issa F., Milward K., Goto R., Betts G., Wood K.J, & Hester J. (2019). Transiently Activated Human Regulatory T Cells Upregulate BCL-XL Expression and Acquire a Functional Advantage in vivo. Frontiers in Immunology, 10, 889.

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