DC were cultured with autologous CD3+ T cells (1:10) for 1 day, with or without the IDO inhibitors 1-MT-D or –L (1 mM, Sigma-Aldrich), where indicated. For immunophenotype studies, tri-color immunofluorescence was performed using fluorescein isotiocyanate (FITC)-conjugated anti-human CD4 (clone RPA-T4), phycoerytrin (PE)-conjugated anti-human Foxp3 (clone 206D) and allophycocyanin (APC)-conjugated anti-human CD25 (clone BC96, Biolegend, San Diego, CA). For cell-surface staining, 1×105 cells/100 µl were incubated in the dark for 20 min at 4°C with mAbs in phosPHAte-buffered saline (PBS)-1% bovine serum albumine. Subsequently, for Foxp3 intracellular staining, cells were incubated at room temperature in the dark for 20 min with fix/perm buffer followed by 15 min with perm solution and additional 30 min with the mAb. After 2 washes, samples were analyzed using BD FACSCanto™II equipment (BD Biosciences). A minimum of 10,000 events was collected in list mode on FACSDiva software. To test their suppressive activity, purified CD4+CD25+ T cells (104/well) were irradiated and added to cultures consisting of CFSE-labeled CD3+ T cells (105/well) as responders, stimulated by 1 µg/ml PHA (Sigma-Aldrich). After 5 days, cultures were analyzed using BD FACSCanto™II equipment (BD Biosciences) and the proliferation index was calculated using FCSExpress4 software.24 (link)
Facscanto 2 equipment
Manufactured by BD
Sourced in United States
The BD FACSCanto II is a flow cytometry system designed for a wide range of applications. It features a compact, benchtop design and utilizes blue and red lasers to detect up to 8 fluorescent parameters simultaneously. The system is equipped with advanced optics and electronics to deliver high-performance data acquisition and analysis capabilities.
Automatically generated - may contain errors
Lab products found in correlation
Paraformaldehyde (pfa), by Merck Group (3 mentions)
Facsdiva software, by BD (3 mentions)
Pe conjugated anti human foxp3 clone 206d, by BioLegend (2 mentions)
Fac scount reagents, by BD (2 mentions)
Tritest trucount monitoring kits, by BD (2 mentions)
Facscanto 2, by BD (1 mentions)
24 well transwell chamber, by Corning (1 mentions)
Carboxyfluorescein succinimidyl ester (cfse), by Merck Group (1 mentions)
1 d mt, by Merck Group (1 mentions)
Allophycocyanin apc conjugated anti human cd25 clone bc96, by BioLegend (1 mentions)
Phytohemagglutinin (pha), by Merck Group (1 mentions)
Anti cd4 fitc clone okt4, by BioLegend (1 mentions)
Brefeldin a, by Merck Group (1 mentions)
Real time hiv 1 amplification matrix, by Abbott (1 mentions)
Cba human th1 th2 th17 cytokine kit, by BD (1 mentions)
Anti human cd45, by BD (1 mentions)
Anti human cd19, by BD (1 mentions)
Anti human cd29, by BioLegend (1 mentions)
Anti human cd90, by Merck Group (1 mentions)
Anti human cd105, by BioLegend (1 mentions)
16 protocols using facscanto 2 equipment
1
Evaluating Treg Suppressive Activity
2
Regulatory T Cell Induction Protocol
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Mo-DCs were cultured with CD3+ T cells (1 : 50) for 5 days with or without 1-MT-L (1 mM). At the end of culture time, cells (1 × 105 cells/100 μL) were incubated with FITC-conjugated anti-human CD4 (clone RPA-T4) and APC-conjugated anti-human CD25 (clone BC96, Biolegend) in the dark for 20 min at 4°C and then for 30 min at room temperature with PE-conjugated anti-human Foxp3 (clone 206D), after 20 min of fixation and 15 min of permeabilization. Samples were analysed using BD FACSCanto II equipment (BD Biosciences). A minimum of 10,000 events was collected in list mode on FACSDiva software.
To test their suppressive activity, at the end of coculture, CD4+CD25+ T cells (104/well) were purified, irradiated, and added to cultures consisting of CFSE-labeled CD3+ T cells (105/well) as responders, stimulated by allogeneic immature Mo-DCs (1 : 10). After 5 days, cultures were analyzed using BD FACSCanto II equipment (BD Biosciences) [25 (link)].
To test their suppressive activity, at the end of coculture, CD4+CD25+ T cells (104/well) were purified, irradiated, and added to cultures consisting of CFSE-labeled CD3+ T cells (105/well) as responders, stimulated by allogeneic immature Mo-DCs (1 : 10). After 5 days, cultures were analyzed using BD FACSCanto II equipment (BD Biosciences) [25 (link)].
3
Modulation of DC Allostimulatory Capacity
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To detect lymphocytes proliferation, CD3+ T cells were labelled with carboxyfluorescein succinimidyl ester (CFSE, 2.5 μM from Sigma Aldrich) before plating [22 (link)]. To test the capacity of Adh-DOC and Det-DOC to modulate the DC allostimulatory capacity, allogeneic Mo-DC, either immature or mature, were irradiated (3000 cGy) and plated (1 : 1) at 37°C on either Adh-DOC or Det-DOC in presence of third party CFSE-labeled CD3+ T cells (1 : 10). As a positive control, CD3+ T cells were cultured with Mo-DC and, as a negative control, with medium alone. For a second set of experiments, CD3+ T cells were added to the upper chamber of a 0.4 μm pore polycarbonate filter in 24-well transwell chambers (Corning Costar) to keep them separated from DC and from either Adh-DOC or Det-DOC, which were plated in the lower chamber of the transwell system. After 5 days, the cells in the upper chamber were collected and analyzed using BD FACSCantoII equipment (BD Biosciences).
4
Immunophenotyping of T Cells and CAR-T Cells
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The BD MultitestTM CD3/CD8/CD45/CD4 kit (Becton, Dickinson, USA) was used according to manufacturer's instructions [31 ]. First, a pipette was used to suck 20 μL BD Multitest CD3/CD8/CD45/CD4 into the bottom of the tube. A 50 μL of cultured T cells and CAR-T cells were transferred to the bottom of the tube and mixed them. The tube was capped and swirled gently for mixing. Tubes were incubated in the dark for 15 minutes at room temperature (20-25°C); then, 450 μL of 1X BD FACS sediment was added to the tube. The tube was capped and swirled gently for mixing. Next, the tube was incubated in the dark for 15 minutes at room temperature (20°C-25°C). Cells were analyzed by using the BD FACS Canto II equipment.
5
Measuring Leukemia-Reactive T Cells
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Leukemia-reactive IFN-γ producing CD3+ T cells were evaluated after the coculture with DCs pulsed with leukemic lysate and matured with the cytokine cocktail containing PGE2. After 4 h of incubation, brefeldin A was added (2 μg/mL, Sigma-Aldrich) and incubated overnight at 37°C. At the end of the incubation, cell-surface staining was performed as described above (anti-CD4 FITC: clone OKT4, anti-CD8 APC: clone HIT8a, Biolegend). Then, cells were fixed (30 min at 4°C in 2% paraformaldehyde (Sigma-Aldrich)) and the anti-IFN-γ antibody (clone B27; Biolegend) was added in 0.1% saponin and incubated for 30 min at 4°C.
Both assays were performed in the presence or absence of 1-MT-L (1 mM). At the end of the culture time, cultures were analyzed using BD FACSCanto II equipment (BD Biosciences).
Both assays were performed in the presence or absence of 1-MT-L (1 mM). At the end of the culture time, cultures were analyzed using BD FACSCanto II equipment (BD Biosciences).
6
Comprehensive HIV Diagnostic Protocol
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Suspected HIV infection was confirmed by qualitative detection of the p24 antigen and anti-HIV-1 and anti-HIV-2 IgG antibodies by enzyme immunoassay (Murex AG/AB Combination Diasorin, UK); serological confirmation was performed using a DPP HIV-1/2 rapid immunoblot kit (Bio-Manguinhos, FIOCRUZ) following the manufacturer's recommendations. The CASA DIA samples did not require complementary diagnostic tests since the institution has its own screening panel, which was used for the enrolled patients.
The plasma HIV viral load was quantified by real-time PCR using the Abbott mSample Preparation System RNA Extraction Kit and the Abbott Real-Time HIV-1 Amplification Matrix (ABBOTT, Chicago, Illinois, USA) following the manufacturer's recommendations.
The quantification of CD4 + T (CD45 high CD3 + CD4 + CD8 -) and CD8 + T (CD45 high CD3 + CD4 -CD8 + ) lymphocytes was performed by immunophenotyping and flow cytometry using BD FACSCalibur-4-color equipment and the FAC-SCountTM reagents and TriTEST™/TruCount monitoring kits (BD Biosciences, San Jose, CA, USA), following the manufacturer's recommendations.
The plasma concentrations of the cytokines IL-17 A, IFN-ɣ, TNF, IL-10, IL-6, IL-4 and IL-2 were determined by cytometric bead array (CBA) using BD FACSCanto™ II equipment and the BD™ CBA Human Th1/Th2/Th17 Cytokine Kit (BD Biosciences, San Jose, CA, USA).
The plasma HIV viral load was quantified by real-time PCR using the Abbott mSample Preparation System RNA Extraction Kit and the Abbott Real-Time HIV-1 Amplification Matrix (ABBOTT, Chicago, Illinois, USA) following the manufacturer's recommendations.
The quantification of CD4 + T (CD45 high CD3 + CD4 + CD8 -) and CD8 + T (CD45 high CD3 + CD4 -CD8 + ) lymphocytes was performed by immunophenotyping and flow cytometry using BD FACSCalibur-4-color equipment and the FAC-SCountTM reagents and TriTEST™/TruCount monitoring kits (BD Biosciences, San Jose, CA, USA), following the manufacturer's recommendations.
The plasma concentrations of the cytokines IL-17 A, IFN-ɣ, TNF, IL-10, IL-6, IL-4 and IL-2 were determined by cytometric bead array (CBA) using BD FACSCanto™ II equipment and the BD™ CBA Human Th1/Th2/Th17 Cytokine Kit (BD Biosciences, San Jose, CA, USA).
7
Multiparametric Lymphocyte Profiling and Cytokine Quantification
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The quantification of T helper (CD45highCD3+CD4+CD8−), T cytotoxic (CD45highCD3+CD4−CD8+), double positive (DP) (CD45highCD3+CD4+CD8+), and double negative (DN) (CD45highCD3+CD4−CD8−) lymphocytes was performed by immunophenotyping and flow cytometry using BD FACSCalibur (4 colors) equipment, FACSCountTM Reagents, and TriTEST™/TruCount monitoring kits (BD Biosciences, San Jose, CA, USA). We used the BD Multiset™ Software v3.1 software (BD Biosciences, San Jose, CA, USA) already standardized for analysis of lymphocyte populations related to HIV infection.
The plasma concentrations of the cytokines IL-17A, IFN-γ, TNF, IL-10, IL-6, IL-4, and IL-2 were determined with a cytometric bead array (CBA) using BD FACSCanto™ II equipment and a BDTM CBA Human Th1/Th2/Th17 Cytokine kit (BD Biosciences, San Jose, CA, USA).
The plasma concentrations of the cytokines IL-17A, IFN-γ, TNF, IL-10, IL-6, IL-4, and IL-2 were determined with a cytometric bead array (CBA) using BD FACSCanto™ II equipment and a BDTM CBA Human Th1/Th2/Th17 Cytokine kit (BD Biosciences, San Jose, CA, USA).
8
Immunophenotyping of Cell Populations
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For immunophenotype studies, dual-color immunofluorescence was performed using the following panel of phycoerythrin (PE)-conjugated or fluorescein isothiocyanate (FITC)-conjugated monoclonal antibodies: anti-human CD13, anti-human CD19, anti-human CD34, anti-human HLA-DR, anti-human CD44, anti-human CD45, anti-human CD73 (Becton Dickinson), anti-human CD14, anti-human CD29, anti-human CD105 (Biolegend), and anti-human CD90 (Chemicon). The cell autofluorescence level was used as the negative control. For cell-surface staining, 1 × 105 cells were incubated, in the presence of the antibodies listed, in PBS/0.5% FBS at room temperature with light protection for 15 min. Cells were rinsed in PBS and analyzed by flow cytometry (FACScanto II equipment; Becton Dickinson). A minimum of 10,000 events was collected in list mode on FACSDiva software.
9
Identifying Bone Marrow Plasma Cells
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At least 100 cells (counted in a Neubauer chamber) of the magnetic cell sorting (MACS) enrichment sample were evaluated by flow cytometry. Anti-CD138-FTIC (B-A38 clone, Exbio), anti-CD38-PE (T16 clone, Immunotech) and anti-CD45-PE-Cy5 (J33 clone, Immunotech) monoclonal antibodies (MoAb) were used to identify bone marrow PCs. Flow analysis was carried out in FACS CantoII equipment (Becton, Dickinson and Company). Data analysis was performed using the BD FACS Diva software (Becton, Dickinson and Company, version 6.1.3, USA) (Figure 2 ).
10
Lysosomal Rupture Assay with Acridine Orange
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BMDMs were loaded with 1 μg/mL Acridine Orange for 20 min (Antunes et al., 2001 (link)). Images were acquired using a Leica SP8 confocal microscope (Leica Microsystems) and data processing was performed with the LAS X Life Science software (Leica Microsystems). Additional samples were acquired in a FACSCanto II equipment (BD Biosciences) and lysosomal rupture was followed by the loss of red fluorescence from the acidic lysosomal compartment in the PerCP-Cy5.5 channel. Data were analyzed by FlowJo Software (Tree Star).
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