The intracellular pluripotent marker OCT4 for iPSCs was characterized via flow cytometry (FCM) using True-NuclearTM Transcription Factor Buffer Set (424401, Biolegend, San Diego, CA, USA). Cells were detached and digested into a single-cell suspension using trypsin. The cells were centrifuged and precipitated in a 15 mL tube. Then, they were fixed with True-NuclearTM 1× for 45 min at room temperature in the dark. We next added 2 mL buffer of True-NuclearTM 1×Perm to the tube, centrifuged the tubes, and discarded the supernatant. Next, we added 100 μL True-NuclearTM 1×Perm buffer and 5 μL PE anti-OCT4 (OCT3) antibody (653703, Biolegend) or the 0.625 μL PE Mouse IgG 2b isotype control (400313, Biolegend) to the samples, which were then incubated at room temperature for 30 min. Cell labeling was analyzed in a staining solution using NovoCyte Quanteon (https://www.agilent.com/en/product/research-flow-cytometry/flow-cytometers/flow-cytometer-systems/novocyte-quanteon-flow-cytometer-systems-4-lasers-984692 , 3 January 2024, Agilent, Santa Clara, CA, USA), and data were analyzed using NovoExpress software (Agilent, America, version 1.6.2).
Novocyte quanteon
Manufactured by Agilent Technologies
Sourced in United States
The NovoCyte Quanteon is a flow cytometry instrument designed for multiparameter analysis of cells and particles. It features advanced technologies to provide high-resolution data and precise quantification of cellular characteristics, including size, granularity, and expression of specific markers.
Automatically generated - may contain errors
Lab products found in correlation
Novoexpress software, by Agilent Technologies (4 mentions)
Biotinylated anti fcεri clone mar 1, by BioLegend (2 mentions)
Anti cd63 pe cy7 antibody clone nvg 2, by BioLegend (2 mentions)
7 aad, by BD (2 mentions)
Mouse fc block, by BD (2 mentions)
Neutravidin, by Thermo Fisher Scientific (2 mentions)
Cell stimulation cocktail, by Thermo Fisher Scientific (2 mentions)
Zombie aqua, by BioLegend (2 mentions)
Anti cd8, by BioLegend (2 mentions)
Kaluza software, by Beckman Coulter (2 mentions)
Attune nxt, by Thermo Fisher Scientific (2 mentions)
Facscanto 2, by BD (2 mentions)
True nuclear transcription factor buffer set, by BioLegend (2 mentions)
Cd34 percp eflour710, by Thermo Fisher Scientific (2 mentions)
Thy 1 pe cy7, by BioLegend (2 mentions)
Fixable live dead nir dead cell stain kit, by Thermo Fisher Scientific (2 mentions)
Cd45 apc cy7, by BioLegend (2 mentions)
P selectin, by BioLegend (1 mentions)
U plex kit, by Mesoscale (1 mentions)
Live dead fixable aqua, by Thermo Fisher Scientific (1 mentions)
44 protocols using novocyte quanteon
1
Characterization of iPSC Pluripotency by FCM
2
Platelet Immunophenotyping in PRP
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Immunophenotyping of platelets in PRP was carried out in a NovoCyte Quanteon™ flow cytometer (Agilent Technologies). The samples were diluted tenfold with phosphate-buffered saline (PBS) and stained with CD61 (557291 BD Biosciences), CD63 (353011 BioLegend) and P-selectin (304910 BioLegend) at ambient temperature for 15 min. The gating strategy was aimed at counting proportions of CD63 and P-selectin positive events in CD61 + pools.
3
Mast Cell Activation and Degranulation Assay
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Human MCs were plated in 96-well round bottom tissue culture plates at 5 × 104 per well and centrifuged for 2 min at 400 g prior to resuspending with anti-FcεRI (clone CRA-1, Miltenyi Biotec) at 250 ng mL−1 plus isotype-control mouse antibody (MOPC21, Allakos Inc) or Siglec-6 mAbs (Allakos Inc) at 5 μg mL−1 or indicated concentration at 4 °C for 2 min. Cells were washed in PBS and then incubated in PBS with 10 μg mL−1 secondary antibody (Thermo). After an additional PBS wash, cells were resuspended and incubated for 20 min at 37 °C for flow analysis or for 6 h for analysis cytokine levels in the supernatant. The percent of CD63 (Biolegend, 353004) and CD107a (Biolegend, 328608) expressing cells was determined by flow cytometry on a Novocyte Quanteon (Agilent). For cytokine quantification, 25 μl of supernatant was analyzed using Meso Scale Discovery’s U-plex KIT (customized for the quantification of the indicated cytokines). Active tryptase activity was determined using the mast cell degranulation assay kit (Millipore Sigma).
4
Siglec-6-Dependent Degranulation Assay
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BMMCs expressing Siglec-6 were plated in 96-well round bottom tissue culture plates at 5 × 104 per well and centrifuged for 2 min at 400 g prior to resuspending with biotinylated anti-FcεRI (clone MAR-1, Biolegend) at 250 ng mL−1 plus biotinylated isotype-control mouse antibody (MOPC21, mouse IgG1, Allakos) or biotinylated Siglec-6 mAb (AK04 mouse IgG1, Allakos) at 5 μg mL−1 at 4 °C for 2 min. Cells were washed in PBS and then incubated in PBS with 10 μg mL−1 neutravidin (Thermo) for 2 min. After an additional PBS wash, cells were resuspended in 200 μl 37 °C complete medium and incubated for 20 min at 37 °C for flow analysis or for 1 or 6 h for analysis of histamine or cytokine levels in the supernatant. For flow cytometry, cells were resuspended in 100 μl cold FACS buffer (PBS/1%BSA) containing 100 ng anti-CD63-PE/Cy7 antibody (clone NVG-2, Biolegend), 3 μl 7-AAD (Becton Dickinson) as viability marker and 0.2 μl mouse Fc block (BD). The percent of CD63 expressing cells was determined by flow cytometry on a Novocyte Quanteon (Agilent).
5
Assessing NK Cell Activation in Cancer
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NCI-H1960, A549 and NCI-H1975 cells were seeded in 6-well plates, incubated overnight and treated with docetaxel and cisplatin (concentrations as described above). After 24 hours of treatment, the appropriate conditions were treated with human aCD70 or isotype control and co-cultured with NK cells at a 5:1 ratio. After washing, cells were stained with the following antibodies: anti-CD45 APC-Cy7 (1:50, Clone 2D1, Biolegend), anti-CD3 PE-Cy7 (1:100, Clone SK7, Biolegend), anti-CD56 PE-CF594 (1:25, Clone NCAM16.2, BD Biosciences), anti-CD69 PerCP-Cy5.5 (1:100, Clone FN50, Biolegend) and Live/Dead Fixable Aqua (1:50, Invitrogen) for 30 minutes at 4°C. Acquisition was performed on the Novocyte Quanteon (Agilent technologies).
6
Multiparametric Flow Cytometry for Immune Cell Profiling
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Single-cell suspensions of in vitro generated DCs or murine tumors, spleens and lymph nodes were incubated with antibodies for 20 min at 4°C, followed by washing with staining buffer (DPBS+2% FBS). Surface staining and intracellular staining for FOXP3 and Ki-67 were performed as previously described.26 (link) IFNγ production was evaluated by intracellular staining after in vitro stimulation with Cell Stimulation Cocktail (eBioscience) for 4 hours, using the intracellular fixation and permeabilization buffer set (eBioscience). Data acquisition was performed on Attune NxT (ThermoFisher) or NovoCyte Quanteon (Agilent) cytometer, and data analyzed by FlowJo software (TreeStar). Antibodies are detailed in online supplemental table S1 .
7
Quantifying Reactive Oxygen Species
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DCFH-DA probe was used to detect ROS generation according to the manufacturer's instruction. In brief, RAW264.7 cells (1 × 106 cells/mL) were seeded in a 12-well plate and incubated overnight. Then, the cells were pretreated with or without CUR, THC and OHC (2, 4, 8 μM) for 2 h before challenged by LPS (100 ng/mL). After 24 h, the cells were added with10 μM DCFH-DA for 30 min in the dark, which was then collected to accurately evaluate ROS production using a fluorescence microscope (Agilent NovoCyte Quanteon, USA) with a multiplate reader at the wavelength of 485 nm and 525 nm (excitation and emission), respectively.
8
Activation of Human Basophils via FcεRI
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S8-BMMC were plated in 96-well round bottom tissue culture plates at 5x104 per well and centrifuged for 2 min at 400g prior to resuspending with biotinylated anti-FcεRI (clone MAR-1, Biolegend) at 250ng/mL plus biotinylated isotype-control mouse antibody (MOPC21, mouse IgG1, Allakos) or biotinylated Siglec-8 mAb (2E2, mouse IgG1, Allakos) at 5μg/mL at 4°C for 2 min. Cells were washed in PBS and then incubated in PBS with 10μg/mL neutravidin (Thermo) for 2 min. After an additional PBS wash, cells were resuspended in 200μl 37°C complete medium and incubated for 20 min at 37°C for flow analysis or for 1 or 6 hours for analysis of histamine or cytokine levels in the supernatant. For flow cytometry, cells were resuspended in 100μl cold FACS buffer (PBS/1%BSA) containing 100ng anti-CD63-PE/Cy7 antibody (clone NVG-2, Biolegend), 100ng anti-CD107a-PE (clone 1D4B, Biolegend), 3μl 7-AAD (Becton Dickinson) as viability marker and 0.2μl mouse Fc block (BD). The percent of CD63 and CD107a expressing cells was determined by flow cytometry on a Novocyte Quanteon (Agilent). For cytokine quantification, 25μl of supernatant was analyzed using Meso Scale Discovery’s U-plex kit (customized for the quantification of the indicated cytokines). Histamine levels were determined using an enzyme immunoassay kit (Beckman Coulter).
9
Evaluating GFP Fusion Constructs in E. coli
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Strains of E. coli TOP10, which were engineered to carry translational fusions of superfolder GFP43 (link) to different variants of the 5′ part of the BT_1675 coding sequence, were cultured in Lysogeny Broth medium supplemented with chloramphenicol (20 μg ml−1) and carbenicillin (100 μg ml−1) until an OD600 of 0.5 was reached. Subsequently, 100 μl of the cultures was collected and subjected to three washes with 1× phosphate-buffered saline before fixation with a 4% paraformaldehyde solution. The fluorescence intensity of GFP was measured in phosphate-buffered saline using flow cytometry (NovoCyte Quanteon; Agilent).
10
Transfection and Flow Cytometry Analysis
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At 72 h, the cells were collected, counted, and resuspended at 2 × 106 cells/mL in fresh media with fresh cytokines and plated in u-bottom 96-well plates in triplicates at 2 × 105 cells/well. The cells were immediately transfected with 600 ng Capstan CD5/LNP-mCherry for 1 h and washed with 1x PBS twice to remove excess tLNP and minimize nonspecific uptake. The transfected cells were then incubated as before for a further 23 h, for a total of 24 h posttransfection.
At 24 h posttransfection, the cells were washed and stained with a viability dye (Zombie Aqua, BioLegend) and labeled with anti-CD3 (pan-T cell marker, BioLegend), anti-CD4 (BD Biosciences), and anti-CD8 (BioLegend) antibodies. The labeled cells were then analyzed on a flow cytometer (Agilent NovoCyte Quanteon, running NovoExpress version 1.6). The data were then analyzed using FlowJo version 10.8.1, and a gating strategy was established to exclude dead cells using the viability dye, followed by gating on CD3+ cells (pan-T cell), and further gating on the two T cell subpopulations CD4+ and CD8+ cells. Last, the expression of mCherry was recorded as a percentage of cells expressing the protein on the gated CD4+ cells and CD8+ cells for each cell culture condition.
At 24 h posttransfection, the cells were washed and stained with a viability dye (Zombie Aqua, BioLegend) and labeled with anti-CD3 (pan-T cell marker, BioLegend), anti-CD4 (BD Biosciences), and anti-CD8 (BioLegend) antibodies. The labeled cells were then analyzed on a flow cytometer (Agilent NovoCyte Quanteon, running NovoExpress version 1.6). The data were then analyzed using FlowJo version 10.8.1, and a gating strategy was established to exclude dead cells using the viability dye, followed by gating on CD3+ cells (pan-T cell), and further gating on the two T cell subpopulations CD4+ and CD8+ cells. Last, the expression of mCherry was recorded as a percentage of cells expressing the protein on the gated CD4+ cells and CD8+ cells for each cell culture condition.
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