At least 10,000 neutrophils were acquired on a three-laser, eight-color BD FACSCanto-IITM flow cytometer (BD Biosciences, San José, CA, USA) and analyzed using BD FACSDiva Software. Overview of lasers for FACSCanto-II TM is presented in S2 Table .
Facscanto iitm
Manufactured by BD
Sourced in United States
The BD FACSCanto II is a flow cytometer designed for multi-parameter analysis of cells. It utilizes laser technology to rapidly analyze and sort fluorescently labeled cells. The system is capable of detecting and measuring various cellular characteristics such as size, granularity, and expression of specific markers.
Automatically generated - may contain errors
Lab products found in correlation
Facsdiva, by BD (2 mentions)
Lipofectamine rnaimax, by Thermo Fisher Scientific (1 mentions)
Annexin 5 allophycocyanin apc, by BioLegend (1 mentions)
Propidium iodide, by Thermo Fisher Scientific (1 mentions)
Annexin 5 binding buffer, by BioLegend (1 mentions)
Fowjo software, by Tree Star (1 mentions)
Cell staining buffer, by BioLegend (1 mentions)
Trustain fcxtm anti mouse cd16 32 antibody, by BioLegend (1 mentions)
Facscanto 2, by BD (1 mentions)
Binding buffer, by BD (1 mentions)
Lysis buffer, by Thermo Fisher Scientific (1 mentions)
Anti cd4 efluor 450 clone rm4 5, by Thermo Fisher Scientific (1 mentions)
Anti foxp3 alexa fluor 488 clone fjk 16s, by Thermo Fisher Scientific (1 mentions)
Fixation buffer, by Thermo Fisher Scientific (1 mentions)
Fitc conjugated lineage cocktail, by BD (1 mentions)
Flowjo7, by Tree Star (1 mentions)
Rnase a, by Merck Group (1 mentions)
Propidium iodide, by Merck Group (1 mentions)
Facsaria 3, by BD (1 mentions)
Rneasy kit, by Qiagen (1 mentions)
18 protocols using facscanto iitm
1
Neutrophil Profiling Using FACSCanto-II
2
miRNA Mimics Transfection in U373MG Cells
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MiRNA mimics (300 ng/well) for UL123 and miR-200b-3p/miR-200c-3p were transiently transfected in U373MG 3′-UTR-SC (1 × 106/well) using Lipofectamine® RNAiMAX (Invitrogen, Carlsbad, CA, USA) in 6-well plates. GFP-positive cells and levels of GFP expression were quantified using flow cytometry (BD FACSCanto IITM, BD Biosciences, San Jose, CA, USA) and immunoblotting, respectively, 2 days after transfection.
3
Apoptosis Profiling of MDA231 Cells
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Cells were arranged at a density of 2.0 × 105 in a 60 mm dish for one tube of flow cytometry. MDA231 single culture or MDA231 cells with astrocyte culture supernatant (50% conditioned medium diluted by DMEM) were seeded at 2.0 × 105 cells/well in a 6-well plate. In the mixed seeding of MDA231 and the astrocyte group, cells were mixed in a 1:1 ratio to obtain a total of 4 × 105 cells/well. After 48 h of culture, the medium was removed and replaced with 120 µM CDDP medium. After an additional 48 h, the cells were trypsinized and washed with PBS. Cells were resuspended in Annexin V binding buffer (BioLegend, San Diego, CA, USA) to 1 × 106 cells/mL. Cell fluid (100 µL) was taken and 5 µL of allophycocyanin (APC) -Annexin V (BioLegend) and 10 µL of propidium iodide (PI) (Invitrogen, Carlsbad, CA, USA) were added, mixed gently, and incubated for another 15 min in the dark. Finally, 400 µL of Annexin V binding buffer was added. The cell fluid was passed through a mesh and transferred to a fluorescence-activated cell sorting tube for flow cytometry using BD FACS Canto IITM (BD Biosciences). For the early apoptotic marker, the Annexin V-positive population was detected, while the late apoptotic marker, PI-positive population, was detected. Finally, Annexin V-positive and PI-negative populations in each culture group were compared.
4
Quantifying Cellular Peptide Internalization
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Cellular internalization of FITC-labelled peptides was analyzed using flow cytometry. Cells were incubated in the presence of the peptides (5 µM each) in complete medium for 1 h. Cells were then washed three times in PBS and incubated with trypsin (1 mg/mL) for 10 min to remove the extracellular unbound peptides. Finally, cells were suspended in PBS and kept on ice. FITC fluorescence intensity of internalised peptides in live cells was measured by flow cytometry using BD FACS CANTO IITM by acquiring 1 × 104 cells. Data was obtained and analysed using FACSDivaTM (BD biosciences) and FowJo software (Treestar Inc., Ashland, Oregon, USA). In some experiments, cellular internalization was analysed using multimode spectrophotometry. Briefly, after incubation with the FITC-labelled peptides, cells were washed as described, centrifuged and the cell pellet resuspended in 300 µL of 0.1 M NaOH. Following 10 min incubation at room temperature, the cell lysate was centrifuged (14,000 × g for 5 min) and the fluorescence intensity of the supernatant determined (494/518 nm). The fluorescence of the cellular uptake is expressed as fluorescence intensity per mg of total cellular protein.
5
Comprehensive Immune Cell Profiling by Flow Cytometry
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For immuno-phenotyping analyses by flow cytometry, isolated peritoneal cells were resuspended in cell staining buffer (Biolegend; 420201), incubated for ten minutes at 4 °C with Tru-Stain fcXTM (anti-mouse CD16/32) antibody (Biolegend; 101320) to prevent non-specific binding, and finally incubated with multiple combinations of fluorescent antibodies for 15 minutes at 4 °C (Table S1 ). Cells were collected on a BD FACSCantoIITM using the BD FACSDIVA Software. For each sample, a total of ten thousand cells were analysed. Data were further analysed using Flow-Jo®.
6
Quantifying DNA Damage and ATM Activation
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MSCs from both experimental groups were incubated with the MuseTMMulti-Color DNA Damage Kit (EDM Millipore Corporation) according to the
manufacturer’s protocol. Briefly, 1X assay buffer was added to the cells,
which were fixed and permeabilized with fixation buffer and permeabilization
buffer, respectively. After washing, they were labeled with two antibodies
(20X anti-phospho-histone H2A.X-PECy5, 20X anti-phospho-ATM-PE) which
measures the phosphorylation status of the histone pH2A.X and the ATM
kinase. Cells were acquired with a BD FACS CantoIITM flow
cytometer and results were obtained using the FlowJoTM 10 software (BD
Biosciences).
manufacturer’s protocol. Briefly, 1X assay buffer was added to the cells,
which were fixed and permeabilized with fixation buffer and permeabilization
buffer, respectively. After washing, they were labeled with two antibodies
(20X anti-phospho-histone H2A.X-PECy5, 20X anti-phospho-ATM-PE) which
measures the phosphorylation status of the histone pH2A.X and the ATM
kinase. Cells were acquired with a BD FACS CantoIITM flow
cytometer and results were obtained using the FlowJoTM 10 software (BD
Biosciences).
7
Measuring Mitochondrial Membrane Potential in BMCs
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JC-1 is a fluorescent dye that measures the mitochondrial membrane potential (ΔΨ) of cells. The loss of this potential serves as an indicator of apoptosis, where this dye remains in its monomeric form and emits a green fluorescence. Living cells form the “J-aggregates” which emit a red fluorescence.
BMCs were treated with 100 μg/mL AEPA for 96 hours. Subsequently, cells were incubated with JC-1 (1 μM) for 30 min at 37°C. After incubation, the cells were washed and resuspended in PBS. Fluorescence data were obtained using a flow cytometer (BD FACSCantoII TM) at an excitation wavelength of 488 nm, where JC-1 monomers emit fluorescence at 529 nm and J-aggregates emit at 590 nm. A total of 10.000 events were acquired for each sample and the data were obtained by flow cytometer BD FACSCantoII. The data were analyzed using WinMDI version 2.9 (Joseph Trotter) software. The gate was determined using unstained BMCs controls (Additional file1 ). The data were analyzed using WinMDI version 2.9 (Joseph Trotter) software.
BMCs were treated with 100 μg/mL AEPA for 96 hours. Subsequently, cells were incubated with JC-1 (1 μM) for 30 min at 37°C. After incubation, the cells were washed and resuspended in PBS. Fluorescence data were obtained using a flow cytometer (BD FACSCantoII TM) at an excitation wavelength of 488 nm, where JC-1 monomers emit fluorescence at 529 nm and J-aggregates emit at 590 nm. A total of 10.000 events were acquired for each sample and the data were obtained by flow cytometer BD FACSCantoII. The data were analyzed using WinMDI version 2.9 (Joseph Trotter) software. The gate was determined using unstained BMCs controls (Additional file
8
Quantifying VACV Infection Efficiency
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To determine the in vitro infection efficiency of double-deleted VACV, 3 × 105 REM134 cells were plated in six-well culture plates and infected with VVTG17990 at MOIs of 10−3, 10−2, 10−1, and 1. Quantitative evaluation of fluorescence was performed 24 h after infection by flow cytometry (BD FACSCanto IITM, BD Biosciences, San Jose, CA, USA). Prior to flow cytometry, single-cell suspensions were fixed with 4% paraformaldehyde (PFA).
9
Apoptosis Analysis via Flow Cytometry
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Apoptosis was examined using flow cytometry with Annexin V-fluorescein isothiocyanate (FITC) and propidium iodide (PI) (cat. no. 556547; BD Biosciences, San Jose, CA, USA). Briefly, cells were treated with various concentrations of gefitinib or cisplatin for 24 h, trypsinized, washed with cold PBS, and suspended in binding buffer (BD Biosciences) at a concentration of 1×107 cells/ml. Thereafter, they were stained with 5 μl of Annexin V-FITC and 5 μl of PI in 100 μl of cell suspension and incubated for 15 min at room temperature in the dark for processing using flow cytometry. All the early apoptotic cells (Annexin V-positive, PI-negative), necrotic/late apoptotic cells (double positive), and living cells (double negative) were counted using FACSCanto IITM (BD Biosciences).
10
Multiparametric Analysis of Lung Immune Cells
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Mediastinal lymph nodes were removed and processed similarly to lung as described in Section “Cytokine Levels in Lung Tissues .” The cells were collected, and the remaining erythrocytes of the left lung were lysed (lysis buffer, eBioscience, San Diego, CA, USA) according to the manufacturer’s instructions. Single-cell suspensions from MLN and lung tissues were resuspended at 1 × 106 cells/mL. The cells were labeled with the following monoclonal fluorochrome-conjugated antibodies: anti-CD3-Brilliant Violet 510 (clone 17 A2, BioLegend, San Diego, CA, USA), anti-CD4-eFluor 450 (clone RM4-5, eBioscience), anti-CD25-APC (clone PC61, BD Pharmigen), anti-OX-40-PE (clone OX-86, eBioscience), anti-PD-1-APC-eFluor 780 (clone J43, eBioscience), and anti-GITR-PE-Cyanine 7 (clone DTLA-1, eBioscience) for 30 min at 4°C in the dark. The cells were then washed in PBS and incubated with a fixation buffer (eBioscience) for 30 min at 4°C in the dark. After washing, the cells were resuspended in permeabilization solution 1× (eBioscience) and stained with anti-FOXP3-Alexa Fluor 488 (clone FJK-16s, eBioscience) for 30 min at 4°C in the dark. Flow cytometry was performed on a FacsCANTO II TM (BD, San Diego, CA, USA). At least 100,000 events were analyzed with FlowJo (Tree Star) or FACSDiva (BD Biosciences) software. Gating strategy is indicated in Figure S2 in Supplementary Material.
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