Single-cell suspensions were acquired from the liver tissues and prepared to measure cellular ROS 2ʹ,7ʹ-dichlorofluorescein diacetate (DCFDA) in all groups by flow cytometry as described previously.23 (link) The samples were acquired on the MACSQuant Analyzer 10 and analyzed using FlowJo software v10.7.
Macsquant analyzer 10
Manufactured by FlowJo
The MACSQuant Analyzer 10 is a compact, automated flow cytometry system designed for cell analysis and sorting. It offers high-performance, multi-color flow cytometry with up to 10 fluorescence channels. The system is capable of analyzing a wide range of sample types, including cells, beads, and microparticles, and provides users with reliable and reproducible data.
Automatically generated - may contain errors
Lab products found in correlation
Dcfda h2dcfda cellular ros assay kit, by Abcam (1 mentions)
Streptavidin percp, by BD (1 mentions)
Propidium iodine, by Thermo Fisher Scientific (1 mentions)
Cd4 fitc clone rm4 5, by Thermo Fisher Scientific (1 mentions)
Mitochondrial membrane potential assay kit, by Abcam (1 mentions)
Annexin 5 fitc pi apoptosis staining kit, by Miltenyi Biotec (1 mentions)
Zombie violet dye, by BioLegend (1 mentions)
Cd3e pe cy7, by BioLegend (1 mentions)
Brefeldin a, by Thermo Fisher Scientific (1 mentions)
Perm wash buffer, by BD (1 mentions)
Apc cy7, by BioLegend (1 mentions)
Percp cy5, by BioLegend (1 mentions)
Zombie violet fixable viability dye, by BioLegend (1 mentions)
B b homodimerizer, by Takara Bio (1 mentions)
Calcein am, by Thermo Fisher Scientific (1 mentions)
11 protocols using macsquant analyzer 10
1
Measuring Cellular Oxidative Stress
2
Intracellular ROS Levels Analysis by Flow Cytometry
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The intracellular ROS levels of living cells were analyzed by the flow cytometry using DCFDA/H2DCFDA-Cellular ROS Assay kit (Abcam, Cambridge, UK), following the manufacturer’s instructions. The cells were divided into seven groups of treatment—namely, G1 (vehicle control of empty liposomes); G2 (+ve control TBHP); G3 (DCPDO IC10); G4 (DCPDD IC10); G5 (DCPDD IC25); G6 (DCPDO IC10 + DCPDD IC10); G7 (DCPDO IC10 + DCPDD IC25). Briefly, 2.5 × 105 cells were grown in 6-well plates for 24 h, treated with selected doses, and incubated for 48 h. The cells were harvested, followed by washing in PBS, and incubated with DCFDA (20 μM), for 30 min at 37 °C. The cells were then acquired using MACSQuant Analyzer 10 and analyzed by FlowJo software v10.8.1. The cells from G2 were exposed to 50 μM of ter-butyl hydrogen peroxide (TBHP), 4 h prior to staining with DCFDA.
3
Multiparametric Flow Cytometry Analysis
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Cells collected from brain, spleen, or BM were filtered through 50-μm cell strainer (Partec) and blocked with antibodies against Fc-receptors (CD 16/32, clone 2.4G2; in house). Cells were stained with following anti-mouse antibodies for the time course experiment: CD3e-PeCy7 (clone 145-2c11; eBioscience), TCRβ-APC (clone H57-597; eBioscience), CD4-FITC (clone RM4-5; eBioscience), CD8-BV570™ (clone 53-6.7; Biolegend), CD49b (clone DX5; in house), and Propidium Iodine (Thermo Fisher Scientific) to test cell viability. The myeloid cell population was stained with the following antibodies: CD45.2-APC, CD11b-Pacific Blue (clone M1/70; in house), Ly6G-FITC (clone 1A8-Ly6g; eBioscience), Ly6C-Biotin (clone AL-21, BD Biosciences), CD115-PE (clone AF598, eBioscience), streptavidin-PerCP (BD Biosciences), and Pacific Orange-N-Hydroxysuccinimide (NHS) (in house) as viability dye. Stained live cells were acquired (stopping gate of 100,000 live cells) using a MACS Quant Analyzer 10 and analyzed by FlowJo software.
4
Mitochondrial Membrane Potential Assay
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The changes in the MMP by FSE at various doses were determined by the flow cytometry as well as the confocal microscopy using TMRE (tetramethylrhodamine, ethyl ester)-Mitochondrial Membrane Potential Assay Kit (Abcam, Cambridge, USA), according to manufacturer’s instructions. TMRE is a cell permeant, positively charged, red-orange dye that readily accumulates in active mitochondria due to their relative negative charge. Depolarized or inactive mitochondria have decreased the membrane potential and fail to sequester. Briefly, 2.5 × 105 cells for flow cytometry and 5 × 104 for confocal image analysis were grown in each well of 6-well and 24 plates, respectively, for 24 hours, followed by the treatment with the designated doses and incubated for 48 hours. The cells were harvested, washed, and incubated with TMRE (200 nM) for 20 minutes at 37°C, followed by acquisition on MACSQuant Analyzer 10 and analysis using FlowJo software v10.7. For confocal image analysis, the TMRE (50 nM) was overlaid to the culture and incubated for 20 minutes. The media, then replaced by PBS and TMRE, was detected under the confocal microscope.
Note: The FCCP (20 µM) was added 10 minutes prior to staining with TMRE in FSE 0 (positive control) treated cells.
Note: The FCCP (20 µM) was added 10 minutes prior to staining with TMRE in FSE 0 (positive control) treated cells.
5
Immune Response Characterization of HCV Peptides
6
Apoptosis Analysis by Flow Cytometry
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The changes in the proportion of cells in the viable, early, and late apoptotic stages were analyzed using Annexin V-FITC/PI Apoptosis Staining Kit (Miltenyi Biotec, Germany). The cells were divided into six groups of treatment—namely, G1 (vehicle control of empty liposomes); G2 (DCPDO IC10); G3 (DCPDD IC10); G4 (DCPDD IC25); G5 (DCPDO IC10 + DCPDD IC10); G6 (DCPDO IC10 + DCPDD IC25). The cells were grown, treated, and harvested, as stated earlier. The cells were then incubated with Annexin V-FITC and PI in binding buffer, at room temperature, and for 15 min in the dark, following the manufacturer’s instructions. The samples were acquired using MACSQuant Analyzer 10 and analyzed by FlowJo software v10.8.1.
7
Flow Cytometry Analysis of Stimulated PMNs
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PMNs were stimulated with SKMEL28 CM, A375 CM, HEMa CM or control medium for 90 min (37 °C, 5% CO2). To determine the viability of the cells (2.5 × 105), Zombie Violet dye (BioLegend, San Diego, CA, USA) was added. The cells were then stained with phosphate buffered saline (PBS) containing 1% FCS for 20 minutes at + 4 °C. Allophycocyanin (APC)-conjugated anti-CD66b (clone REA306, 1:50), VioBlue-conjugated anti-CD193 (clone REA574, 1:10), PerCP-conjugated anti-CD11b (clone REA713, dilution 1:50) and FITC-conjugated anti-CD62L (clone 145/15, dilution 1:50) were used, all from Miltenyi Biotech (Bergisch Gladbach, Germany). The MACS Quant Analyzer 10 and FlowJo software version 10 were used to analyze the results. Doublets and debris (identified based on forward and side scatter properties), dead cells (identified using the Zombie Violet Fixable Viability Kit) and eosinophils (identified based on the CCR3+ exclusion gate) were excluded from the analysis. All the experiments were run in duplicate.
8
Cytokine Production Assay for Malaria Peptides
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3 × 106 isolated leukocytes were distributed in 96-well plates and resuspended in 100 μl of complete RPMI medium. The immunogenic peptides PbGAP5040−48 (SQLLNAKYL-NH2; peptides&elephants, Hennigsdorf, Germany) (22 (link)) and PbTRAP130−138 (SALLNVDNL-NH2; peptides&elephants, Hennigsdorf, Germany) (51 (link)) were used. 50 μl peptide diluted in RPMI (10 g/ml) was incubated for 2 h at 37°C, 5% CO2. Thereafter, 50 μl of Brefeldin A (1:1,000; eBioscience) was added to block secretion of cytokines, and cells were incubated for additional 4 h. After centrifugation cells were stained for surface markers with CD3e-PeCy7, CD4-BV421™ (clone RM4-5; Biolegend) and CD8-BV570™ antibodies, followed by staining with Pacific Orange-NHS for cell viability. Cells were fixed with 4% paraformaldehyde for 15 min at room temperature, and then permeabilized with Perm/Wash™ buffer (BD Biosciences). Intracellular IFN-γ was stained with Interferon gamma-APC antibody (clone XMG 1.2; eBioscience). Cells were washed in Perm/Wash™ buffer, then resuspended in PBS/1% BSA for acquisition (stopping gate of 20,000 CD8+ T cells) with MACS Quant Analyzer 10 and analysis by FlowJo software (51 (link)).
9
Macrophage Identification and Viability
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PECs isolated from experimental animals were stained and incubated for 30 min with Zombie Violet Fixable Viability Dye (BioLegend) to assess dead cells. After incubation, cells were washed with PBS, and the Fc receptor of the cells was blocked using an anti-mouse CD16/32 antibody (Fcγ R III/II, Ly-17; BioLegend). After 30 min of incubation at 4°C, cells were washed with FACS Buffer (PBS containing 1% FBS) and subsequently centrifuged at 1,200 rpm for 5 min. After removing the supernatant, cells were resuspended in 100 µL of antibody master mix and incubated for 30 min at 4°C. The following antibodies were used in this experiment: F4/80 (PerCP-Cy5.5 BioLegend) and CD11b (APC-Cy7, BioLegend). Unlabeled or isotype-matched stained cells were used as controls. Dead cells were excluded, and macrophages were identified as F4/80+ CD11b+. The acquisition was performed using a Miltenyi MACSQuant Analyzer 10 instrument, and the data were analyzed using FlowJo software (FlowJo, LLC).
10
Quantifying IL-6 in Murine Sera and B-GCs
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IL-6 concentration determination was performed on the sera of experimental mice and on the supernatants of differentiated B-GCs at 48 h using the commercial assay The BD™ CBA Mouse IL-6 Flex Set (Cytometric Bead Array, BD Biosciences, cat. 5622236). The samples were read using a MACSQuant Analyzer 10 flow cytometer and data were analyzed with FlowJo 10 software.
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