MitoSox Red (Thermo Scientific, Cat. M36008), a highly selective mitochondrial superoxide indicator for live cells, was used to measure ROS levels. Rotenone (200 nM, Sigma), known to be a mitochondrial superoxide inducer, was used as a positive control. To account for superoxidespecific fluorescence, the cells were pretreated with 100 units/mL PEG-SOD (Sigma) or 20 μM MnP for 24 h prior to measurement. In brief, cells were loaded with 5 μM MitoSox Red for 10 min at 37 °C and rinsed three times with HBSS and then collected by trypsinization. Cell suspension (final volume of 500 μL in 1× PBS) was analyzed using a BD FACS LSR II flow cytometer (Becton Dickinson). Ten thousand cells were acquired for each sample using the FACS DIVA software (Becton Dickinson). The results were then analyzed using Cell Quest Pro software.
Facs lsr 2 flow cytometer
Manufactured by BD
Sourced in United States
The BD FACS LSR II™ is a flow cytometer designed for analyzing and sorting cells. It utilizes laser technology to detect and measure various characteristics of cells passing through a fluid stream. The core function of the BD FACS LSR II™ is to provide quantitative data about the physical and biochemical properties of cells within a sample.
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Lab products found in correlation
Facsdiva software, by BD (8 mentions)
Anti icos pe, by BD (4 mentions)
Anti cd127 fitc, by Thermo Fisher Scientific (4 mentions)
Anti cd25 apc cy7, by Thermo Fisher Scientific (4 mentions)
Live dead fixable dead cell stain kit, by Thermo Fisher Scientific (4 mentions)
Red blood cell lysis buffer, by BioLegend (3 mentions)
Peg sod, by Merck Group (2 mentions)
Mitosox red, by Thermo Fisher Scientific (2 mentions)
Rotenone, by Merck Group (2 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (2 mentions)
Fluorophore conjugated antibodies, by BD (1 mentions)
Accutase treatment, by Thermo Fisher Scientific (1 mentions)
Facscalibur flow cytometer, by BD (1 mentions)
Apc brdu flow kit, by BD (1 mentions)
Dq ova, by Thermo Fisher Scientific (1 mentions)
Synergy htx plate reader, by Agilent Technologies (1 mentions)
Rnase a, by Beyotime (1 mentions)
16 protocols using facs lsr 2 flow cytometer
1
Mitochondrial Superoxide Measurement in Cells
2
Measuring Mitochondrial Superoxide with MitoSox
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MitoSox Red (Thermo Scientific, Cat. M36008), a highly selective mitochondrial superoxide indicator for live cells, was used to measure ROS levels. Rotenone (200 nM, Sigma), known to be a mitochondrial superoxide inducer, was used as a positive control. To account for superoxide-specific fluorescence, the cells were pretreated with 100 units/mL PEG-SOD (Sigma) or 20 μM MnP for 24 h prior to measurement. In brief, cells were loaded with 5 μM MitoSox Red for 10 min at 37 °C and rinsed three times with HBSS and then collected by trypsinization. Cell suspension (final volume of 500 μL in 1× PBS) was analyzed using a BD FACS LSR II flow cytometer (Becton Dickinson). Ten thousand cells were acquired for each sample using the FACS DIVA software (Becton Dickinson). The results were then analyzed using Cell Quest Pro software.
3
Isolation and Characterization of Immune Cells
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Epididymal white adipose tissue (epiWAT) was excised, minced, and digested with collagenase type II, and the stromal vascular fraction isolated as described previously. Spleens were isolated, homogenized and suspended in PBS. Bone marrow derived cells were collected by flushing the femur and tibia with PBS. These cells were centrifuged at 500 × g for 5 min. Whole blood was centrifuged at 500 × g, 4°C for 5 min and plasma was collected. The remaining blood cells and the resulting pellets were re-suspended in 1X red blood cell lysis buffer (Biolegend, San Diego, CA), at room temperature for 3 minutes followed by addition of 1 X PBS and centrifugation. Then, blood cells, spleen cells and bone marrow derived cells were stained with anti-CD11b, anti-7/4 and anti-Gr-1, stromal vascular fraction was stained with anti-CD11c and F4/80, both followed by incubation at room temperature for 45 minutes. Cells were subsequently washed with 1 X PBS and re-suspended in 1% neutral buffered formalin and run by flow cytometry (BD FACS LSR II™ flow cytometer, Becton Dickinson, San Jose, CA). Data was analyzed using BD FACS Diva software (Becton Dickinson, San Jose, CA). All antibodies were purchased from Biolegend, Miltenyi Biotec, or BD Bioscience.
4
Isolation and Characterization of Immune Cells
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VAT from the mice were excised, minced, and digested with collagenase type II, and the stromal vascular fractions cells were isolated as described previously [30 (link)]. Bone marrow derived cells were collected by flushing the femur and tibia with PBS. These cells were centrifuged at 500 x g for 5 min. Whole blood was centrifuged at 500 x g, 4 °C for 5 min and plasma was collected. The remaining blood cells and the resulting pellet of stromal vascular fraction cells were re-suspended in 1X red blood cell lysis buffer (Biolegend, San Diego, CA), at room temperature for 3 min followed by addition of 1 X PBS and centrifugation. Then, blood cells and bone marrow cells were stained with anti-CD11b, anti-7/4 and anti-Ly6G, stromal vascular fraction cells were stained with anti-CD11C, CD206 (MR) and F4/80, both followed by incubation at room temperature for 45 min. Cells were subsequently washed with 1 X PBS and re-suspended in 1% neutral buffered formalin and run by flow cytometry (BD FACS LSR II™ flow cytometer, Becton Dickinson, San Jose, CA). Data was analyzed using BD FACS Diva software (Becton Dickinson, San Jose,CA). All antibodies were purchased from Biolegend, Miltenyi Biotec, or BD Bioscience.
5
In Vitro Hematopoietic Differentiation
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10 human HSCs per well (20–60 wells per donor) were sorted for differentiation analysis in 96-well plates seeded with MS5-cells and medium containing α-MEM supplemented with 10% FCS, 2-ME, gentamycin, rhSCF (10 ng/ml), hFLT3-L (10 ng/ml), rhIL-3 (5 ng/ml), and IL-7 (5 ng/ml). Following seeding, 50% of medium was exchanged with fresh medium containing all cytokines as above, except rhIL-3, once a week. Following 5 weeks of culture, cells were incubated with Al700 anti-human CD45 (HI30), APC anti-human CD11b (ICRF44) and CD33 (WM53), PerCy7 anti-human CD19 (HIB19), all from BioLegend (San Diego, US), as well as APCe780 anti-human CD3 (SK7; eBioscience, San Diego, CA). Propidium Iodine was used to exclude dead cells. Cell analysis was performed on a BD FACS LSRII flow cytometer using FACSDiva software (Becton Dickinson, Franklin Lakes, NJ), and further analyzed using FlowJo software (Treestar, Ashlad, OR).
6
Isolation and Characterization of Immune Cells
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Visceral adipose tissues from the mice were excised, minced, and digested with collagenase type II, and the SVF isolated as described previously.These cells were centrifuged at 500 × g for 5 min. Whole blood was centrifuged at 500 × g, 4°C for 5 min and plasma was collected. The remaining blood cells and the resulting pellets were re-suspended in 1X red blood cell lysis buffer (Biolegend, San Diego, CA), at room temperature for 3 minutes followed by addition of 1 X PBS and centrifugation. Then, blood cells spleen cells and bone marrow derived cells were stained with anti-CD11b, anti-7/4 and anti-Gr-1, SVFs were stained with anti-CD11c and F4/80, both followed by incubation at room temperature for 45 minutes. Cells were subsequently washed with 1 X PBS and re-suspended in 1% neutral buffered formalin and run by flow cytometry (BD FACS LSR II™ flow cytometer, Becton Dickinson, San Jose, CA). Data was analyzed using BD FACS Diva software (Becton Dickinson, San Jose, CA). All antibodies were purchased from Biolegend, Miltenyi Biotec, or BD Bioscience [23 (link),53 (link)].
7
Macrophage Phenotype Characterization
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The levels of surface markers of differentiation (CD11b or CD14) and polarization (HLA-DR and CD80 for M1 or CD206 and CD163 for M2a and c phenotypes) were assessed in THP-1 cells/PBMs and TDMs/MDMs of different phenotypes. Selected markers of M1 (HLA-DR) and M2 (CD206) phenotypes were analysed in MDMs treated with 500 nM NMU-9 (for 24 h) using immunofluorescence staining and flow cytometry. Macrophages were detached on ice with cold 0.5% BSA in PBS with 2 mM EDTA, resuspended in PBS/0.5% BSA/2 mM EDTA and stained with fluorophore-conjugated antibodies (all from BD Biosciences, Franklin Lakes, NJ, USA, described in Additional file 2 : Table S1) for 30 min at RT in the dark. A washing step with PBS containing 2 mM EDTA was performed. The FACS analysis was performed using a FACS LSR II BD flow cytometer (Becton Dickinson) equipped with BD FACS Diva Software. The results were analysed using FlowJo™ 10.7.1 software (BD).
8
Antibody Staining and Flow Cytometry
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Cells were detached by accutase treatment (Thermo Fisher Scientific), resuspended in PBS / 1 % BSA / 2 mM EDTA and stained with fluorophore-conjugated antibodies for 30 min at RT in the dark or unconjugated antibodies for 45 min at RT. Washing step with PBS with 2 mM EDTA was performed. When needed, cells were stained with corresponding secondary antibodies conjugated to AlexaFluor 488 (Thermo Fisher Scientific) for 30 min at RT in the dark with following washing step (PBS). Antibodies details are collected in the Table S3 (Additional File 1 ). FACS analysis was performed using FACS LSR II BD flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA) equipped with BD FACSDiva Software. The results were analysed by FlowJo ™ 10.7.1 software (BD).
9
Quantification of TLR2 Expression
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Mtb-infected and noninfected macrophages, as well as phagocytes treated with TBChoD recombinant protein, were detached from plates and suspended in Dulbecco's phosphate-buffered saline (D-PBS) containing 1% FBS. To prevent nonspecific anti-TLR2 antibody binding, we blocked the crystallisable fragment receptors (FcRs) in D-PBS supplemented with 10% human AB serum for 15 minutes at room temperature. Next, cells were washed twice in D-PBS/1% FBS and stained with 10 μg/ml of a PE-conjugated mouse anti-TLR2 monoclonal antibody (Novus Biologicals) or with a PE-conjugated mouse IgG2a isotype control (Becton Dickinson, Franklin Lakes, USA) for 30 minutes at 4°C, or the cells remained untreated. Next, all of the samples were washed twice in D-PBS/1% FBS and then examined with a FACS LSR II BD flow cytometer (Becton Dickinson) equipped with BD FACSDiva Software. The results were analysed by WinMDI software, and the data are presented as the median fluorescence intensity (MFI), which correlates with the surface expression of the target molecule.
10
Analyzing Cell Cycle Dynamics with Flow Cytometry
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Growth curves were performed by daily counting of viable pre-miR-183 transfected and scramble transfected cells expressing GFP protein using an LSRII FACS flow cytometer (BD Biosciences Europe, Erembodegem, Belgium). For cell cycle distributions, pre-miR-183 transfected and scramble transfected cells were collected and incubated 1 h at 4°C with propidium iodide (0.05 mg/ml) solution containing Non-idet-P40 (0.05%) for proliferation and cell cycle assays. Cells were analyzed using a FACSCalibur flow cytometer (BD Biosciences Europe, Erembodegem, Belgium) and cell cycle distribution was determined using Modfit LT 2.0™ software (Veritysoftware Inc., Topsham, ME, USA). BrdU incorporation assays were performed using the APC-BrdU flow kit (BD Pharmingen), following the manufacturer’s protocol.
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