Counting of Th17, Treg, exhausted T, and exhausted Treg cell was performed using flow cytometry. The monoclonal antibodies of surface and intracellular antigens used for counting Th17 cells were CD4-FITC (BioLegend, US, cat. no. 357405) and IL-17A-PE (BioLegend, US, cat. no. 506903), and CD4-FITC (BioLegend, US, cat. no. 357405), CD25-PE (BioLegend, US, cat. no. 985802), and CD127-PerCP-Cy5.5 (BioLegend, US, cat. no. 351321) were used for Treg cells; CD8-FITC (BioLegend, US, cat. no. 980908), PD-1-PerCP-Cy5.5 (BioLegend, US, cat. no. 329913), and Tim-3-PE (BioLegend, US, cat. no. 345006) for exhausted T cells; and CD4-FITC (BioLegend, US, cat. no. 357405), CD25-PE (BioLegend, US, cat. no. 985802), and PD1-PerCP-Cy5.5 (BioLegend, US, cat. no. 329913) for exhausted Treg cells [18 (link)].
Tim 3 pe
Manufactured by BioLegend
Sourced in United States
Tim-3-PE is a fluorescent-conjugated antibody that binds to the T-cell immunoglobulin and mucin domain-containing protein 3 (Tim-3). Tim-3 is a cell surface receptor that plays a role in the regulation of immune responses.
Automatically generated - may contain errors
Lab products found in correlation
Cd8 fitc, by BioLegend (4 mentions)
Cd25 pe, by BioLegend (4 mentions)
Pd 1 percp cy5, by BioLegend (2 mentions)
Cd4 fitc, by BioLegend (2 mentions)
Pd 1 pe, by BD (2 mentions)
Pd1 apc, by BioLegend (2 mentions)
Cd3 fitc, by BioLegend (2 mentions)
Cd127 apc, by BioLegend (2 mentions)
Facsverse, by BD (2 mentions)
Lag 3 pe, by BioLegend (2 mentions)
Cd127 percp cy5, by BioLegend (1 mentions)
Il 17a pe, by BioLegend (1 mentions)
Cd3 bv570, by BioLegend (1 mentions)
Foxp3 buffer set, by BD (1 mentions)
Eomes efluor 660 wd1928, by Thermo Fisher Scientific (1 mentions)
Cd4 bv605 rpa t4, by BD (1 mentions)
Cd8 bv650 rpa t8, by BD (1 mentions)
Live dead near ir, by Thermo Fisher Scientific (1 mentions)
Lsrii flow cytometer, by BD (1 mentions)
Cd8 bv650, by BioLegend (1 mentions)
12 protocols using tim 3 pe
1
Quantification of Immune Cell Subsets
2
Comprehensive Immunophenotyping of T Cells
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Cryopreserved peripheral blood mononuclear cells were thawed and stained in Horizon Brilliant Stain Buffer (BD) containing all antibodies and Live/Dead Near-IR (Life Technologies) at 1:300 dilution and stained at 4°C for 30 minutes in Horizon. Panel 1 included the following: CD3 BV570 (UCHT1), CCR7 Pacific Blue (G043H7), and CD27 AlexaFluor700 (M-T271) (all BioLegend); CD4 BV605 (RPA-T4) and CD8 BV650 (RPA-T8) (BD); programmed cell death protein 1 (PD-1) phycoerythrin (PE)–eFluor610 (eBioJ105), CD45RA fluorescein isothiocyanate (HI100), and T cell immunoreceptor with immunoglobulin and ITIM domains (TIGIT) peridinin-chlorophyll protein complex (PerCP)–eFluor710 (MBSA43) (eBioscience); and Tim-3 PE (344823) (R&D).
Panel 2 included the following: CD3 BV570, CD38 AlexaFluor700 (HB-7) (BioLegend); CD4 BV605, CD8 BV650, PD-1 PE-eFluor610, and Tim-3 PE. After this, cells were washed twice before fixation and permeabilization with Foxp3 Buffer Set (BD). Staining for intracellular epitopes was performed with: T-bet fluorescein isothiocyanate (4B10) (BioLegend) and Eomes eFluor660 (WD1928) (eBioscience). Samples were acquired on an LSR II flow cytometer (BD). Data were analyzed using FlowJo software (version 10.8.0r1; Tree Star).
Panel 2 included the following: CD3 BV570, CD38 AlexaFluor700 (HB-7) (BioLegend); CD4 BV605, CD8 BV650, PD-1 PE-eFluor610, and Tim-3 PE. After this, cells were washed twice before fixation and permeabilization with Foxp3 Buffer Set (BD). Staining for intracellular epitopes was performed with: T-bet fluorescein isothiocyanate (4B10) (BioLegend) and Eomes eFluor660 (WD1928) (eBioscience). Samples were acquired on an LSR II flow cytometer (BD). Data were analyzed using FlowJo software (version 10.8.0r1; Tree Star).
3
Flow Cytometry Analysis of CAR T Cell Phenotype
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Expression of EGFR and other proteins on the tumor cell surface was detected by flow cytometry using the anti-EGFR antibody (BD, San Jose, CA, USA). In brief, the CAR T cells were collected from culture and assayed with monoclonal antibodies against human CD25-PC5 and CD69-PC5 (Beckman Coulter Inc., Indianapolis, IN, USA), CD3-PE-CY5, CD4-PE, CD8-FITC, CD45RO-PE-CY5, CD62L-PE, TIM3-PE, and LAG3-Alexa Fluor 647 (Biolegend, San Diego, CA, USA) and CCR7-FITC, CD107α-PE-CY5, and PD-1-PE (BD, San Jose, CA, USA) according to a previous study40 (link) or the manufacturers’ instructions.
EGFR-modified CAR expression was detected by using an indirect method with biotinylated EGFR protein and streptavidin-coupled PE antibody (BD). Fluorescence was assessed using a Beckman Coulter Gallios™ flow cytometer, and the data were analyzed with the FlowJo vX.0.7 and Kaluza v1.5 software (Beckman Coulter Inc.).
EGFR-modified CAR expression was detected by using an indirect method with biotinylated EGFR protein and streptavidin-coupled PE antibody (BD). Fluorescence was assessed using a Beckman Coulter Gallios™ flow cytometer, and the data were analyzed with the FlowJo vX.0.7 and Kaluza v1.5 software (Beckman Coulter Inc.).
4
Comprehensive Immune Phenotyping Protocol
5
Profiling Exhausted CD4+ T Cells
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Phenotypic markers expressed by CD4+ T cells isolated at day 0 and from day 14 were analysed by flow cytometry. Cells were fixed using Human FoxP3 Buffer Set (BD Biosciences, 560098), and stained with anti-human: CD4 BV510, LAG-3 Pe/Cy7, TIM-3 PE, TIGIT PeDazzle594, CTLA-4 BV421 (all BioLegend, 317444, 369310, 345006, 372716, 369606), CD3 FITC, and PD-1 APC (both ThermoFisher, 11-00390-42, 17-2799-42 respectively). Cells were then analysed using a cyAn ADP flow cytometer (Beckman Coulter).
6
Phenotypic Analysis of Engineered Lymphocytes
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The studied cells were freshly stained with anti-MUC1 antibody (EP1024Y) diluted to 1:50 in 2% FBS in PBS. Antibodies that were used to investigate the cell phenotypes including anti-CD3-FITC, CD4-APC, CD8-APC, CD19-APC, CD16-APC, CD69-APC, and CD62L-APC were purchased from ImmunoTools GmbH (Friesoythe, Germany), anti-CD45RA-PE-cyanine7 and PD-1-PE from Invitrogen (Carlsbad, USA), anti-CD56-PE, LAG-3-PE and TIM-3-PE from BioLegend (San Diego, USA), and anti-CD25-PerCP-cyanine5.5 from eBioscience, Inc. (California, USA).
To detect the CARs expression on transduced lymphocytes, the transduced cells were harvested, blocked with 1% BSA, and stained with biotin-conjugated protein-L (Thermo Fisher Scientific, Waltham, MA, USA). Alexa Fluor 488 dye-conjugated streptavidin was finally added. The data were acquired on a BD FACSVerse or BD Accuri C6 flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA) and analyzed with FlowJo software version 10.
To detect the CARs expression on transduced lymphocytes, the transduced cells were harvested, blocked with 1% BSA, and stained with biotin-conjugated protein-L (Thermo Fisher Scientific, Waltham, MA, USA). Alexa Fluor 488 dye-conjugated streptavidin was finally added. The data were acquired on a BD FACSVerse or BD Accuri C6 flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA) and analyzed with FlowJo software version 10.
7
HIV-1 Infection Inhibition Assay
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PBMCs (1 x 106/ml) were infected with 100 TCID50 of various HIV-1 strains (92UG037: primary R5-tropic strain; YU2B and BaL: R5-tropic lab strains, 93BR020: dual-tropic primary isolate; NL4-3: X4-tropic lab strain) and simultaneously treated with a range of concentrations of IND02-trimer in 24-well plates (Greiner) in duplicates. Cells were washed, fixed (4% paraformaldehyde) and stained on several days post infection (1-4-9-13-19) using the following mAbs against PD-1-Brilliant Violet 421, CD3-FITC, Tim-3-PE, CD4-PE-Cy7 (all from Biolegend) and CD8-APC (Miltenyi). Multicolor flow cytometric analyses were performed on a FACS Verse (BD Biosciences).
8
Isolation and Analysis of PBMCs
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In order to isolate peripheral blood mononuclear cells (PBMCs) from each sample, 1 mL of heparinized whole blood from CHB patients or healthy controls was collected and subjected to Ficoll-Hypaque density gradient centrifugation. After centrifugation, PBMCs were collected and washed with FACS buffer (phosphate-buffered saline [PBS] containing 0.5% BSA, BD Biosciences). For T-cell staining, the collected PBMCs were stained with the following fluorochrome-conjugated anti-human antibodies: CD3-APC, CD8-FITC, PD-1-PE, Tim-3-PE, CD3-APC, CD4-PerCP and CD25-FITC (Biolegend, San Diego, CA, USA) for 30 min. The stained cells were then washed with FACS buffer (BD Biosciences) and resuspended in a Fixation/Permeabilization Solution (BD Biosciences), prior to being washed and resuspended with PBS, and acquired on a FACSCalibur flow cytometer (BD Biosciences). After gating, the proportions of CD3+CD8+PD-1+, CD3+CD8+Tim-3+ and CD3+CD4+CD25high cells were determined. The data were analyzed using FlowJo software (Tree Star, Ashland, OR, USA).
9
Multiparameter Flow Cytometry Immunophenotyping
10
Comprehensive Immune Profiling of CAR T Cells
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CAR T cells (2×105 cells per test) were labeled with antibodies at 4°C for 15 min. Flow cytometry was performed using a MACSQuant Analyser 10 Flow Cytometer (Miltenyi Biotec, Bergisch Gladbach, Germany), and data were analyzed by FlowJo V.10.7.1 software (FlowJo). Cells were subjected to SSc-FSc and gated as the CD3+ T-cell population. For transfection efficiency: CD19 CAR detection reagent, anti-biotin PE, and CD3-APC (Miltenyi Biotec, Bergisch Gladbach, Germany); T-cell phenotype: CD3-PerCP (UCHT1), CD8-PE (SK1), (BD Bioscience, New Jersey, USA), CD4-APC (BioLegend, San Diego, California, USA); memory phenotype: CD3-PerCP (UCHT1), CD45RO-VioGreen (UCHL1), and CD62L-VioBlue (DREG-56) (BD Bioscience, New Jersey, USA); exhaustion: CD3-PerCP, PD1(CD279)-APC, TIM-3-PE, LAG3-PE, and TIGIT-APC (BioLegend, San Diego, California, USA); activation: CD3-PerCP, CD25-PE, and CD69-APC (BioLegend, San Diego, California, USA); and for cytolytic activity: CD3-PE (OKT3), CD19-APC (HIB19) (BioLegend, San Diego, California, USA), and 7AAD-PerCP (BD Bioscience, New Jersey, USA).
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