On day 7, the tumour-bearing mice were treated with 3 mg/kg SZU-101 or 4 mg/kg DOX by intraperitoneal administration. Mouse spleens were collected at 2, 4 or 24 h post treatment on day 8, and the splenocytes were prepared by removing the red blood cells with RBC lysis buffer (BioLegend) after separating the cells through a 70-μm cell strainer. Approximately 1 × 106 cells were stained with corresponding florescence antibodies and analysed by FACSCalibur flow cytometry (BD Biosciences). The antibodies for flow cytometry were purchased from BioLegend and included the following: anti-CD11c-Alexa488, anti-CD86-PerCP/Cy5.5, anti-CD80-Alexa647, anti-CD3-Alexa488, anti-CD19-Alexa647 and anti-CD69-PerCP/Cy5.5.
Anti cd69 percp cy5
Manufactured by BioLegend
The Anti-CD69-PerCP/Cy5.5 is a fluorescently labeled antibody that targets the CD69 cell surface marker. CD69 is a type II membrane protein and is commonly used as an early activation marker for various immune cell types. The PerCP/Cy5.5 fluorophore is attached to the antibody, allowing for detection and analysis of CD69-expressing cells using flow cytometry.
Automatically generated - may contain errors
Lab products found in correlation
Anti ifn γ apc antibody, by BioLegend (2 mentions)
Anti cd8 pe cy7, by BD (2 mentions)
Anti cd25 pe, by BioLegend (2 mentions)
Anti cd86 percp cy5, by BioLegend (1 mentions)
Anti cd3 alexa488, by BioLegend (1 mentions)
Facscalibur flow cytometry, by BD (1 mentions)
Rbc lysis buffer, by BioLegend (1 mentions)
Anti cd11c alexa488, by BioLegend (1 mentions)
Brefeldin a, by BioLegend (1 mentions)
Anti cd8 brilliantviolet605, by BioLegend (1 mentions)
Anti cd3 fitc, by BioLegend (1 mentions)
Anti cd4 pe cy7, by BioLegend (1 mentions)
Ebioscience foxp3 transcription factor staining buffer set, by Thermo Fisher Scientific (1 mentions)
Anti cd107a apc cy7, by BioLegend (1 mentions)
Migg1 af488, by BioLegend (1 mentions)
Anti cd8 bv510, by BioLegend (1 mentions)
Anti pd 1 percpcy 5, by BioLegend (1 mentions)
Anti cd25 bv421, by BioLegend (1 mentions)
Canto 2, by BD (1 mentions)
Anti cd8 pe cy7, by BioLegend (1 mentions)
6 protocols using anti cd69 percp cy5
1
Immunomodulatory Effects of SZU-101 and Doxorubicin
2
Cytokine Profile and NK Cell Activation Assay
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Healthy human PBMCs were seeded at 0.5 million cells/ml in RPMI (10% FBS, 1% Pen/Strep) in a 24-well plate. TLR agonists were incubated with the PBMCs overnight at 1 μM. The supernatant was collected and analyzed for cytokine concentrations. Target cells were trypsinized and added to the wells at an effector to target ratio of 2:1. Cetuximab was added at a final concentration of 200 nM. Anti-CD107a APC Cy7 (Biolegend, San Diego, CA) was added to all samples. The samples were incubated at 37 °C. One hour later, Brefeldin A (Biolegend, San Diego, CA) was added and the samples were once again incubated at 37 °C. Four hours later, cells were collected and stained for extracellular markers using anti-CD3 FITC, anti-CD56 Brilliant Violet 650, anti-CD4 PE-Cy7, anti-CD8 Brilliant Violet 605, and anti-CD69 PerCP-Cy 5.5 (Biolegend, San Diego, CA) antibodies. Intracellular staining was performed with anti-IFN-γ APC antibody (Biolegend, San Diego, CA) using eBioscience Foxp3 Transcription Factor Staining Buffer Set (Thermofisher, Waltham, MA). All samples were set up in duplicates. Samples were analyzed using a BD Fortessa H0081 flow cytometer at the University of Minnesota Flow Cytometry Core Facility.
3
Multiparametric Flow Cytometry for Immune Cell Analysis
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Human cells were stained with the following fluorochrome-labeled antibodies: anti-CD45-PECy7 (Biolegend), anti-CD8-BV510 (Biolegend), anti-CD25-BV421 (Biolegend), anti-CD69-PerCPCy5.5 (Biolegend), anti-CD137-Biot (5D1, the laboratory’s own hybridoma), anti-Ki67-AF488 (Biolegend) and anti-Granzyme-AF647 (Biolegend). Streptavidin-PE was added to detect 5D1-Biot Ab. Isotype control mixes was prepared with mIgG1-BV421 (Biolegend), mIgG1-PerCPCy5.5 (Biolegend), mIgG1-AF488 (Biolegend), mIgG1-AF647 (Biolegend). In the case of 5D1-Biot, FMO was performed. M11 CAR expression was verified with anti-mIgG(H+L)-AF647 (Invitrogen) just before stimulation with mesothelin-Fc-beads.
Mouse T cells were stained with: anti-CD8-PECy7 (Biolegend), anti-CD25-APC (Biolegend), anti-CD69-BV510 (Biolegend), anti-CD137-PE (Biolegend), anti-PD1-PerCPCy5.5 (Biolegend), and anti-Ki67-AF488 (BD Bioscience). Rat IgG1-APC (Biolegend), Arm Hamster-BV510 (Biolegend), Syr Hamster-PE (Biolegend), Rat IgG2a-PerCPCy5.5 (Biolegend) and mIgG1-AF488 (Biolegend) antibodies were used as isotype-matched negative controls.
Zombie NiR (Biolegend) was used to exclude cell death. Samples were acquired on a BD Canto II (BD Biosciences) and CytoFlex S systems (Beckman Coulter). Analyses were performed using FlowJo (Tree Star) and CytExpert software (Beckman Coulter).
Mouse T cells were stained with: anti-CD8-PECy7 (Biolegend), anti-CD25-APC (Biolegend), anti-CD69-BV510 (Biolegend), anti-CD137-PE (Biolegend), anti-PD1-PerCPCy5.5 (Biolegend), and anti-Ki67-AF488 (BD Bioscience). Rat IgG1-APC (Biolegend), Arm Hamster-BV510 (Biolegend), Syr Hamster-PE (Biolegend), Rat IgG2a-PerCPCy5.5 (Biolegend) and mIgG1-AF488 (Biolegend) antibodies were used as isotype-matched negative controls.
Zombie NiR (Biolegend) was used to exclude cell death. Samples were acquired on a BD Canto II (BD Biosciences) and CytoFlex S systems (Beckman Coulter). Analyses were performed using FlowJo (Tree Star) and CytExpert software (Beckman Coulter).
4
Evaluating NK Cell Activation
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PBMCs were seeded (5 × 105 cells/well) in 96-well
U-bottom plates and treated with the compounds (1 μM) or vehicle
(0.1% DMSO) for 24 h. Anti-CD107a FITC and monensin (Biolegend, San
Diego, CA) were added to all wells for the last 4 h of incubation.
The cells were washed and stained with Live/Dead Fixable Aqua Dead
Cell Stain (Invitrogen, Carlsbad, CA). After further washing, Fc receptors
were blocked with Human TruStain FcX (Biolegend) and cells were stained
for extracellular markers using anti-CD3 APC/Fire 750, anti-CD56 PE,
and anti-CD69 PerCP-Cy5.5 antibodies (Biolegend). Intracellular staining
was performed with an anti-IFN-γ APC antibody (Biolegend) after
fixation and permeabilization using the Cyto-Fast Fix/Perm Buffer
Set (Biolegend). Samples were analyzed using an Attune NxT flow cytometer
(Thermo Fisher Scientific, Waltham, MA) and the FlowJo software (Tree
Star, Inc., Ashland, OR). Following exclusion of dead cells, CD3- CD56+ NK cells were evaluated for expression
of CD107a, CD69, and IFN-γ. The gating strategy is described
in detail inFigure S6 . Statistical significance
was determined with one-way ANOVA with subsequent Bonferroni’s
multiple comparisons test.
U-bottom plates and treated with the compounds (1 μM) or vehicle
(0.1% DMSO) for 24 h. Anti-CD107a FITC and monensin (Biolegend, San
Diego, CA) were added to all wells for the last 4 h of incubation.
The cells were washed and stained with Live/Dead Fixable Aqua Dead
Cell Stain (Invitrogen, Carlsbad, CA). After further washing, Fc receptors
were blocked with Human TruStain FcX (Biolegend) and cells were stained
for extracellular markers using anti-CD3 APC/Fire 750, anti-CD56 PE,
and anti-CD69 PerCP-Cy5.5 antibodies (Biolegend). Intracellular staining
was performed with an anti-IFN-γ APC antibody (Biolegend) after
fixation and permeabilization using the Cyto-Fast Fix/Perm Buffer
Set (Biolegend). Samples were analyzed using an Attune NxT flow cytometer
(Thermo Fisher Scientific, Waltham, MA) and the FlowJo software (Tree
Star, Inc., Ashland, OR). Following exclusion of dead cells, CD3- CD56+ NK cells were evaluated for expression
of CD107a, CD69, and IFN-γ. The gating strategy is described
in detail in
was determined with one-way ANOVA with subsequent Bonferroni’s
multiple comparisons test.
5
Expansion and Characterization of CMV-specific CD8+ T Cells
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Peripheral blood mononuclear cells (PBMC) were isolated by Ficoll gradient centrifugation from heparinized blood of healthy volunteers. PBMCs were peptide-pulsed (25 mg/mL), washed, and expanded. Media were supplemented with IL2 (20 U/mL, Roche), IL7, and IL15 (both 25 ng/mL, Peprotech). Cells were restimulated every two weeks with freshly prepared or thawed autologous peptide-pulsed PBMCs. These PBMCs were irradiated and washed before use. Interleukins as described above were added. Cells were expanded every 2 to 3 days with fresh medium supplemented with interleukins. Effector-to-target cell ratio (E:T) was calculated for the total expansion culture containing about 50% expanded CMV-pp65-specific CD8 þ T cells and the remaining irradiated PBMCs. PBMCs were washed with PBS/2% FCS and stained with anti-CD8 PE-Cy7 (BD Biosciences), Dextramer CMV APC (Immudex), anti-CD25 PE (BioLegend), or anti-CD69 PerCP/Cy5.5 (BioLegend) on ice for 45 minutes, washed with PBS/2% FCS, and analyzed in FACSCanto II.
6
Measuring T Cell Activation and Downregulation
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In vitro-expanded T cells containing 50% of CMV-pp65peptide-specific CD8 þ T cells were incubated with CMV-single pMHCI-bivalent IgG and double pMHCI-bivalent IgG containing either one or two pMHCI complexes, respectively, at concentrations of 50 nmol/L and 500 nmol/L. After 16 hours, cells were stained with anti-CD8 PE-Cy7 (BD Biosciences), HLA-A Ã 0201-CMV-pp65 (NLVPMVATV)-specific Dextramer (Immudex), anti-CD25 PE (BioLegend), or anti-CD69 PerCP Cy5.5 (BioLegend) to measure downregulation of TCR and activation of T cells in FACSCanto II.
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