PBMC were labeled for 40 minutes at room temperature with 4 cocktails of fluorescently conjugated antibodies (targets, clones, and suppliers are listed in Table E1 ) in PBS 1% BSA, washed in PBS 1% BSA and then PBS alone, and stained with live/dead fix aqua (Invitrogen). Stained cells were fixed in 4% paraformaldehyde (ThermoFisher) and analyzed using a LSRII (BD Bioscience) or iQue3 (Sartorius) flow cytometer. Data were gated using FlowJo version 10 (BD Bioscience) or ForeCyt version 7.02 (Sartorius). Gating strategies are shown in Fig. E7 .
Ique3
Manufactured by Sartorius
Sourced in Germany
The IQue3 is a high-throughput flow cytometry system developed by Sartorius. It is designed to automate the analysis of cells and particles, enabling rapid and efficient data collection. The IQue3 features advanced fluidics and optics to provide accurate and reliable measurements, making it a versatile tool for a wide range of applications in life science research and development.
Automatically generated - may contain errors
Lab products found in correlation
Forecyt version 7, by Sartorius (2 mentions)
Live dead fix aqua, by Thermo Fisher Scientific (2 mentions)
Paraformaldehyde (pfa), by Thermo Fisher Scientific (2 mentions)
Reconstituted guinea pig complement, by Cedarlane (1 mentions)
Gelatin veronal buffer, by Merck Group (1 mentions)
Prism 9, by GraphPad (1 mentions)
7 protocols using ique3
1
Multicolor Flow Cytometry of PBMCs
2
Complement-Mediated Immune Complex Assay
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ADCD was assessed as described previously.21 (link) Immune complexes were formed by incubating plasma samples diluted 1:10 in PBS and coupled biotinylated antigen with 1.0-μm red fluorescent neutravidin beads (Thermo Fisher Scientific) for 2 hours at 37°C in 96-well plates per antigen, in duplicate. The beads were washed with PBS and incubated with reconstituted guinea pig complement (Cedarlane Labs) diluted in gelatin veronal buffer (Sigma-Aldrich) for 20 minutes at 37°C. The reaction was then stopped with 15 mM EDTA in PBS. Goat anti–guinea pig fluorescein isothiocyanate (FITC)-conjugated C3 polyclonal antibody (MP Biomedicals) diluted 1:100 in PBS was used to detect C3 deposition on the beads by flow cytometry (iQue 3; Sartorius). The median fluorescence intensity (MFI) of all bead-positive events in the FITC channel was analyzed with ForeCyt 8.1. Results were visualized with GraphPad Prism 9.
3
OX40 Receptor Binding Assay
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WT IgG, iAbaff1, or hexameric versions of the anti-OX40 clone, 3C8, were labeled with pHAb amine reactive dye according to the manufacture’s protocols (Promega, cat#G9841). OX40 expressing Jurkat cells were treated with the indicated concentration of each pHAb labeled format for 1 h at 37 °C and 5% CO2 in RPMI media containing 10% FBS and 2 mM L-glutamine (cRPMI). Cells were then washed twice with PBS containing 1% BSA and fluorescence was measured using the BL2 channel of a Sartorius iQue3.
4
Multicolor Flow Cytometry of PBMCs
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PBMC were labeled for 40 minutes at room temperature with 4 cocktails of fluorescently conjugated antibodies (targets, clones, and suppliers are listed in Table E1 ) in PBS 1% BSA, washed in PBS 1% BSA and then PBS alone, and stained with live/dead fix aqua (Invitrogen). Stained cells were fixed in 4% paraformaldehyde (ThermoFisher) and analyzed using a LSRII (BD Bioscience) or iQue3 (Sartorius) flow cytometer. Data were gated using FlowJo version 10 (BD Bioscience) or ForeCyt version 7.02 (Sartorius). Gating strategies are shown in Fig. E7 .
5
Vaspin-Induced Apoptosis in Granulosa Cells
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Firstly, 1 × 10 5 granulosa lutein cells were seeded in a 12-well plate. Next, the cells were cultured with different concentrations of Vaspin (80, 160, 320 ng/ml) for 24 h, followed by a Annexin V-FITC/PI staining (BL110A, Biosharp, China). The suspended cells were collected in a 1.5 ml centrifuge tube, centrifuged at 4 °C and 1000 g for 5 min, and resuspended twice with PBS. Subsequently, 3 × 10 5 granulosa lutein cells were resuspended with 500 μL of Anneixn-FITC buffer, and the resuspension solution was incubated with 5 μL of Annexin V-FITC and propidium iodide (PI) for 5 min after mixing them evenly. The binding of the granulosa lutein cells to Annexin V-FITC/PI was then analyzed using high-throughput flow cytometry (iQue® 3, Sartorius, Germany); the binding of the cells to PI was observed at an excitation wavelength of 488 nm and an emission wavelength of 520 nm; and the binding of the cells to Annexin V-FITC was observed at an excitation wavelength of 535 nm and an emission wavelength of 617 nm.
6
Quantification of Adenosine Receptor Expression
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Cells were seeded in 96-well plates at 100,000 cells per well and pelleted at 1200rpm for 4 min. Cells were resuspended in blocking solution (100uL PBS+0.5% BSA per well) and incubated for 1 hr at 4°C. After blocking, cells were pelleted again, resuspended in 100 nM antibody in PBS+0.5% BSA, and incubated for 1 hr at 4°C. Cells were washed twice with PBS+0.5% BSA and then incubated in secondary antibody (Jackson ImmunoResearch #115-606-071 [anti-mouse] and #109-135-098 [anti-human]) in PBS+0.5% BSA for 30 min at 4°C. After two more washes, cells were resuspended in PBS+0.5% BSA and run on the Invitrogen Attune or Sartorius iQue3 flow cytometers. The following antibodies were used as positive controls for each adenosine receptor: Anti-Human Adenosine A1 Antibody (Proteintech #20332-I-AP), Anti-Human Adenosine A 2A R Antibody (R&D #MAB9497), Anti-human Adenosine A 2B R antibody (Sigma-Aldrich #SAB2500030), Anti-Human Adenosine A 2A R/A 2B R Antibody (R&D, #MAB94972), Anti-Human Adenosine A3 Antibody (Invitrogen #PA5-85704), and Anti-Human Adenosine A 2A R Antibody (which cross-reacts with mouse A 2A R; Sigma-Aldrich, #05-717).
7
Cell Surface Receptor Analysis
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For analysis of cell surface receptors, cells were run on the Sartorius iQue3 instrument based on the manufacturer’s instructions. Briefly, 2–5 × 105 cells were dispensed in 100 μl aliquots and blocked with 300 μg/ml hIgG on ice for 1 h. Primary antibodies (5 μg/ml) were then added for 1 h on ice, followed by the addition of labeled secondary detection antibodies as needed. After washing with PBS buffer, the cells were analyzed for fluorescence intensity. Irrelevant rabbit or human IgG was used as negative control.
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