300,000 siRNA transfected DAOY cells per well were seeded 24 h after transfection in low adhesion six well plate (657970; Greiner Bio-one) and incubated for 6 h in SFM. Cells were then treated with either pre-warmed SFM or SFM supplemented with 20 ng/ml HGF for 30 min. After stimulation, cells were fixed by adding paraformaldehyde pre-warmed to 37°C to a final concentration of 4% and incubated for 20 min at 37°C. After one wash with PBS supplemented with 2% FBS, cells were stained with PE-labelled anti-human CD155 (337610, 1:300; BioLegend), corresponding isotype control antibody (400114, 1:300; BioLegend), or left unstained for 20 min on ice. Samples were washed with PBS supplemented with 2% FBS, resuspended in PBS and acquired using BD LSRFortessa flow cytometer (BD Bioscience). Fluorescence levels were analyzed using FlowJo software (BD Bioscience).
Isotype control antibody
Manufactured by BioLegend
Sourced in United States
Isotype control antibodies are laboratory reagents used to establish background staining levels in flow cytometry and other immunoassays. They are antibodies of the same isotype as the target-specific antibody, but they do not bind to any cellular antigen. Isotype controls are essential for distinguishing specific from non-specific antibody binding.
Automatically generated - may contain errors
Lab products found in correlation
Lsrfortessa flow cytometer, by BD (2 mentions)
Facscalibur, by BD (2 mentions)
Cytofix cytoperm solution, by BD (2 mentions)
Percoll, by Cytiva (2 mentions)
Cellquest software, by BD (2 mentions)
Avidin percp, by BioLegend (2 mentions)
Flow cytometry, by Beckman Coulter (1 mentions)
Cd44 fitc, by BioLegend (1 mentions)
Cd90 fitc, by BioLegend (1 mentions)
Cd29 pe, by BioLegend (1 mentions)
Cd45 fitc, by Santa Cruz Biotechnology (1 mentions)
Cd34 fitc, by Santa Cruz Biotechnology (1 mentions)
Dab substrate kit, by Agilent Technologies (1 mentions)
Accustain trichrome stains masson, by Merck Group (1 mentions)
Rat anti mouse mac 2 antibody, by Cedarlane (1 mentions)
Mouse ifn γ, by Miltenyi Biotec (1 mentions)
Incucyte s3 live imaging system, by Sartorius (1 mentions)
Incucyte cytotox green dye, by Sartorius (1 mentions)
Incucyte nuclight rapid red dye, by Sartorius (1 mentions)
Perm buffer 3, by BD (1 mentions)
45 protocols using isotype control antibody
1
Quantification of CD155 Expression in DAOY Cells
2
Phenotypic Characterization of NPSCs
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NPSCs were trypsinised, collected and washed twice with phosphate-buffered saline (PBS). Then, the NPSCs were stained with the following conjugated antibodies: CD29-PE (Biolegend), CD90-FITC (Biolegend), CD44-FITC (Biolegend), CD45-FITC (Santa Cruz) and CD34-FITC (Santa Cruz). An isotype control antibody (Biolegend) was used for each examined antibody. The final antibody concentration was 1 mg/100 mL. After incubating in the dark for 30 min at 37 °C, the cells were washed three times with PBS. Samples were subjected to flow cytometry (Beckman, USA), and the percentage of positive staining was calculated relative to the isotype control staining.
3
Aortic Root Cryosection Analysis
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Serial 10 μm cryosections were cut distally from the aortic root. Sections were stained (200, 400, and 600 µm after the appearance of the aortic cusps) with Oil Red O (Sigma-Aldrich, St. Louis, MO, USA) and Masson´s trichrome (Accustain Trichrome Stains–Masson; from Sigma-Aldrich). For immunohistochemical staining of macrophages, aortic root cryosections (160 μm from the aortic cusps) were incubated with rat anti-mouse Mac-2 antibody (1:1000; Cedarlane, Hornby, BC, Canada) or isotype control antibody (1:1000; Biolegend, San Diego, CA, USA), followed by horseradish peroxidase–conjugated goat anti-rat IgG secondary antibody (1:1000; GE Healthcare, London, United Kingdom) and visualized with the DAB substrate kit (Dako, Glostrup, Denmark). The areas of Oil red O staining, Mac-2 staining, and collagen staining (blue color in Masson’s trichrome) were determined using morphometric analysis (BioPix Software) and normalized to the size of the atherosclerotic lesion. Lesion complexity (presence/absence of necrotic core) was evaluated in sections stained with Masson´s trichrome (200 μm from the aortic cusps).
4
MHC-II Blockade on Tumor Cytotoxicity
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To achieve an effector-to-target (E:T) ratio of 10:1, 10,000 Tsc2-deficient 105K cells were plated in 96 well plates, pre-treated with 10 ng/ml of mouse IFN-γ (Miltenyi Biotech, Cat#130-105-774), and Incucyte Nuclight Rapid Red Dye (1:500 dilution, Sartorius, Cat#4717) for 48 hr before addition of tumor-infiltrating T cells. Each well contained 100 ul of medium supplemented with 1 µl/well of Incucyte Cytotox Green Dye (Sartorius, Cat#4633). For MHC-II blocking experiments, 10 µg/ml of isotype control antibody (BioLegend, Cat#400601) or MHC-II antibody (BioLegend, Cat#107601) was added to each well pre-plated with Tsc2-deficient 105K cells and cultured for 2 hr before addition of CD45+CD3+CD4+CD25-CD38+CD39+ TILs. Cell culture was monitored by the IncuCyte S3 Live Imaging system (Sartorius) at 1-hr intervals for up to 48 hr when needed. All experiments were carried out with samples from each group pooling from 10 independent tumors. The number of dying tumor cells was determined by co-localization of the Cytotox Green signal to tumor cells. Any out of focus frames were discarded.
5
Intracellular Co-localization of KLRG1 and p-AMPK
6
Modulating PD1 Pathway in PBMC
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Freshly isolated PBMCs were cultured in 24-well plates at 2 × 106 cells/well in RPMI 1640 containing 1% penicillin/streptomycin (Sigma), L-Glutamine and 10% heat-inactivated human serum, with or without γ-Mtb CFP-10 + ESAT-6 (10 μg/ml) at 37 °C in a humidified 5% CO2 atmosphere. To neutralize PD1, 10 μg/ml anti-PD1 antibody (BioLegend) was added to the cells in presence of the antigen. Isotype control antibody μg/ml (BioLegend) was added to some cells this served as a control well for PD1 specific inhibition. After 96 h, cell-free culture supernatants were collected, aliquoted and stored at − 70 °C until cytokine concentrations were measured by ELISA as published in [8 (link)]. Cells were washed and stained for surface and intracellular markers.
7
Immunomodulatory Effects of Cytokines
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Recombinant rat interleukin 4 (IL-4) and granulocyte-macrophage colony-stimulating factor (GM-CSF) were purchased from Peprotech (Rocky Hill, NJ, USA). RPMI-1640 medium, Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were from Hyclone (Logan, UT, USA). Phycoerythrin (PE)-conjugated anti-CD80, -CD86, and -MHC-II, Alexa Fluor 647-CD103 and isotype control antibody were purchased from BioLegend (San Diego, CA, USA). The antibodies against β2-AR, GRK2 and β-actin were from Abcam (Cambridge, UK). Horseradish peroxidase (HRP)-labeled goat anti-rabbit and goat anti-mouse antibodies were acquired from Santa Cruz Biotechnology (CA, USA). LPS, FITC-dextran (40 kD), isoprenaline hydrochloride, CGP20712A, ICI118551 and CCK-8 were purchased from Sigma (St. Louis, MO, USA). Salbutamol sulfate was from Shanghai Xudong Haipu Pharmaceutical Co., Ltd. (Shanghai, China). ELISA kits for interleukin 10 (IL-10) and TNF-α were purchased from RayBiotech, Inc. (Norcross, GA, USA).
8
Isolation and Culture of Endothelial Cells
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Primary human dermal blood vascular endothelial cells (BEC) and lymphatic endothelial cells (LEC) were isolated from neonatal human foreskins as previously described [32] . BECs were cultured in endothelial basal medium (EBM; Lonza), supplemented with 20% fetal bovine serum (FBS; Invitrogen), 2 mM L-glutamine, antibiotic-antimycotic solution (Invitrogen), and 10 μg/ml hydrocortisone (Sigma). LECs were cultured in the same medium with additional N6,2-O-dibutyryladenosine-3′,5′-cyclic monophosphate (25 μg/mL; Sigma). Cells were used at passage 6. For FACS analyses, BECs and LECs were stained with PE-labeled anti-human CXCR4 (eBioscience) or isotype control antibody (BioLegend).
9
CD8+ T Cell Activation Assay
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48 well plates (Corning) were coated with either 1.5 μg/mL anti-CD3 (Biolegend) and 5 μg/mL Isotype control antibody, 1.5 μg/mL anti-CD3 and 5 μg/mL PD-L1 (Biolegend), or 1.5 μg/mL anti-CD3 and 5 μg/mL PD-L2 (Biolegend) for 16 hours at 4°C. Mononuclear cells were isolated from whole blood of healthy donors by Lymphoprep density gradient (Stemcell). CD8 T cells were then purified by CD8 Microbeads (Miltenyi). Prior to stimulation, 5 x 105 cells per condition were stained with 2 μM CellTrace Far Red proliferation dye (Thermo) for 20 minutes at 37°C protected from light. Excess proliferation dye was neutralized with warm media, the stained cells were washed in PBS, resuspended in enriched media, and then added to the appropriate stimulation wells at 0.5 mL per well. Cells were incubated for 4 days at 37°C with 5% CO2. On day 4 the cells were analyzed for proliferation dye intensity and surface protein expression by flow cytometry as described below.
10
Anti-CD20 Antibody Mediated B Cell Depletion
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