The following antihuman antibodies were used for cell staining: CD3-PECy7 or CD3-AF700, CD4-FITC, CD8-APC, CD45-PE, CD56-PE, iNKT TCR (Vα24-Jα18 TCR)-APC, CD45RA-FITC, CD62L-PECy7, TIM3-FITC, PD1-APC, LAG3-PECy7, HLA-A2-FITC, HLA-A3-APC, HLA-B7-PECy7, and Annexin V-FITC or Annexin V-Pacific Blue and were purchased from BioLegend. Data acquisition was performed using either a BD Accuri C6 Flow cytometer (BD Biosciences) or an Attune NXT cytometer (Thermo Fisher). Flow cytometry data were analyzed using FlowJo software (Tree Star).
Tim 3 fitc
Manufactured by BioLegend
Sourced in United States
TIM-3-FITC is a fluorescently-labeled antibody that specifically binds to the TIM-3 (T-cell immunoglobulin and mucin-domain containing-3) receptor. TIM-3 is an inhibitory receptor expressed on various immune cells, including T cells, natural killer cells, and dendritic cells. The FITC (Fluorescein Isothiocyanate) label allows the detection and visualization of TIM-3-expressing cells using flow cytometry or other fluorescence-based techniques.
Automatically generated - may contain errors
Lab products found in correlation
Ccr7 percp cy5, by BD (3 mentions)
Lag 3 pe, by BioLegend (2 mentions)
Cd4 apc h7, by BD (2 mentions)
Cd8a pe cy7, by BD (2 mentions)
Cd45ra apc, by BioLegend (2 mentions)
Cd28 fitc, by BioLegend (2 mentions)
Cxcr3 af488, by BD (2 mentions)
Tigit percp cy5, by BioLegend (2 mentions)
Recombinant fc tagged bcma protein, by Enzo Life Sciences (2 mentions)
Bv421 conjugated anti fc antibody, by BioLegend (2 mentions)
Cd27 pe, by BioLegend (2 mentions)
Cd8a fitc, by BioLegend (2 mentions)
Facscanto 2, by BD (2 mentions)
Fc block, by BD (2 mentions)
Cd3 bv421, by BD (2 mentions)
Cd56 pe, by BioLegend (1 mentions)
Cd8 apc, by BioLegend (1 mentions)
Cd4 fitc, by BioLegend (1 mentions)
Annexin 5 fitc, by BioLegend (1 mentions)
Cd45 pe, by BioLegend (1 mentions)
6 protocols using tim 3 fitc
1
Multicolor Flow Cytometry Immunophenotyping
2
Phenotyping of CAR-T Cell Surface Markers
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Cell surface proteins on CAR-T cells were stained with the following antibodies: CD4-APC/H7, CD8a-PE/Cy7, CCR7-PerCP/Cy5.5, CXCR3-AF488, CD69-FITC, CD3-APC (all from BD Biosciences, San Jose, CA, USA); CD8a-FITC, CD28-FITC, CD27-PE, LAG-3-PE, TIM-3-FITC, TIGIT-PerCP/Cy5.5, CD45RA-APC (all from Biolegend, San Diego, CA, USA); PD-1-APC (eBioscience, Thermo Fisher Scientific, San Diego, CA, USA). To determine CAR expression, cells were labeled with a recombinant Fc-tagged BCMA protein (Enzo Life Sciences, Farmingdale, NY, USA) followed by a BV421-conjugated anti-Fc antibody (Biolegend). Samples prepared from mouse organs were treated with Fc block (BD Biosciences) prior to staining. Cells were washed and resuspended in 1% (v/v) FCS in PBS or 1% (v/v) paraformaldehyde prior to acquisition. Live cells were gated based on forward and side scatter.
For intracellular staining of γH2AX, surface-stained cells were fixed in 1% (v/v) paraformaldehyde, permeabilized in 90% (v/v) methanol at −20 °C overnight, and then stained with PE-conjugated anti-H2AX (pS139) antibody (BD Biosciences). Immediately prior to analysis, cells were washed and resuspended in 1% (v/v) FCS in PBS containing 3.3 µg/mL 7-AAD.
All flow cytometry data were acquired on a FACSCanto II (BD Biosciences) and analyzed using FlowJo software, version 7.6.2 (TreeStar, Ashland, OR, USA).
For intracellular staining of γH2AX, surface-stained cells were fixed in 1% (v/v) paraformaldehyde, permeabilized in 90% (v/v) methanol at −20 °C overnight, and then stained with PE-conjugated anti-H2AX (pS139) antibody (BD Biosciences). Immediately prior to analysis, cells were washed and resuspended in 1% (v/v) FCS in PBS containing 3.3 µg/mL 7-AAD.
All flow cytometry data were acquired on a FACSCanto II (BD Biosciences) and analyzed using FlowJo software, version 7.6.2 (TreeStar, Ashland, OR, USA).
3
Comprehensive Profiling of T Cell Markers
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Cell surface proteins on (CAR-)T cells were stained with the following antibodies: annexin V-PE, CCR7-PerCP/Cy5.5, CD3-allophycocyanin (APC), CD3-BV421, CD4-APC/H7, CD8a-PE/Cy7, CD25-PE/Cy7, CD69-fluorescein isothiocyanate (FITC), CD95-PE, CD127-PerCP/Cy5.5, CXCR3-AF488 (all from BD Biosciences, San Jose, CA); CD8a-APC, CD8a-FITC, CD27-PE, CD28-FITC, CD45RA-APC, LAG-3-PE, TIGIT-PerCP/Cy5.5, TIM-3-FITC (all from BioLegend, San Diego, CA); and PD-1-APC (eBioscience, Thermo Fisher Scientific). Monocytes were stained with CD14-PE (eBioscience). To determine CAR expression, cells were labeled with a recombinant Fc-tagged BCMA protein (Enzo Life Sciences, Farmingdale, NY) followed by a BV421-conjugated anti-Fc antibody (BioLegend). Samples prepared from mouse organs were treated with Fc block (BD Biosciences) prior to staining. Cells were washed and resuspended in fluorescence-activated cell sorting (FACS) buffer (1% FCS in PBS) or 1% paraformaldehyde prior to acquisition. In experiments to assess viability and/or apoptosis, cells were resuspended in FACS buffer containing 1 μg/mL 7-aminoactinomycin D (7-AAD). In all other experiments, live cells were gated based on forward and side scatter.
All flow cytometry data were acquired on a FACSCanto II (BD Biosciences) and analyzed using FlowJo software version X.0.7 (Treestar, Ashland, OR).
All flow cytometry data were acquired on a FACSCanto II (BD Biosciences) and analyzed using FlowJo software version X.0.7 (Treestar, Ashland, OR).
4
Flow Cytometry Analysis of T Cell Phenotype
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For surface staining, in vitro cultured human T cells or NCG mice-retrieved PBMCs were stained for 30 min at 4 °C with the following antibodies at 1:200 dilution: CD8a PE/Cyanine7 (BioLegend, 344711), PD-1 PB (BioLegend, 329915), TIM-3 FITC (BioLegend, 345021), and CD39 PE (BioLegend, 328207). Intracellular staining was performed as described above and the flowing antibodies were used at 1:200 dilution: TNFα APC (BioLegend, 502913), IFNγ FITC (BioLegend, 502505), IL-2 PE (BioLegend, 500306), Ki-67 PerCP/Cyanine5.5 (BD Pharmingen, 561284), and Granzyme-B AF647 (BioLegend, 515405). Samples were run on a BD LSRFortessa and data were analyzed using FlowJo (10.4).
5
Comprehensive PBMC Characterization by Flow
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For surface staining, PBMCs from liquid nitrogen were thawed and washed twice in phosphate buffered saline containing 1% fetal bovine serum (staining buffer). Cells were incubated with directly conjugated monoclonal antibodies for 30 min at 4 °C. The cells were then washed and resuspended in staining buffer before flow cytometric analysis. The monoclonal antibodies used were anti-human CD3-PerCp-Cy5.5 or CD3-BV421, CD4-FITC or CD4-V500, CD8-APC-H7, CD45RA-PE-Cy7, CCR7-PerCp-Cy5.5 (BD Biosciences, San Diego, CA, USA), PD-1-PE, TIM-3-FITC, 2B4-APC, BTLA-BV421, CD160-PE-Cy7 (BioLegend, San Diego, CA, USA) and LAG-3-AF700 (R&D Systems, Minneapolis, MN, USA) antibodies, and corresponding isotype controls. Data acquisition was performed on a LSR Fortessa flow cytometer (BD Biosciences) and data analysis was performed using FlowJo Software (Tree Star, Ashland, OR, USA).
6
Evaluating Tumor Cell Surface Markers
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Cells (BTSC233, JHH520, NCH644) were cultivated in 12-well flat-bottom plates (500,000 cells in 2 mL total volume) in the presence of 1 µM drug studied (AE2G3, temozolomide, dasatinib) in standard conditions for 48 h. Non-treated cells were used as a control (non-treated control, NTC). Cells were then collected and washed twice by cold PBS (Gibco) containing 10% FBS (fetal bovine serum, Gibco) and 2 mM EDTA (ethylenediaminetetraacetic acid, Sigma-Aldrich, Taufkirchen, Germany). For evaluating the expression of tumor cells’ surface markers, we used monoclonal antibodies conjugated with fluorochromes: TIM-3-FITC (Biolegend, Amsterdam, the Netherlands), PD-L1-PE (Biolegend, Amsterdam, the Netherlands), and CD47-APC (BD, Eysins, Switzerland). To distinguish dead and live cells, Annexin V-Pacific Blue antibodies (Biolegend, Amsterdam, the Netherlands) were used. Flow cytometry analysis was done on a CyAn (Daco, Beckman Coulter, Denver, CO, USA) cell analyser using Summit (Beckman Coulter, Denver, CO, USA) and Flow Jo (BD, Eysins, Switzerland) software. Experiments were performed using three independent biological repetitions before statistical analysis.
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