Reactive oxygen species (ROS) were detected by the DCF method. Briefly, 8×104 cancer cells were cultured in a 24-well plate overnight. Cells were loaded with 10 μM 2′, 7′-dichlorofluorescin diacetate (DCFDA; Sigma-Aldrich, St. Louis MO, USA) with or without 10 μg exosomes for 2 h at 37°C. A positive and negative control were also set up using 10 mM NAC and 250 μM H2O2, respectively. Cells were acquired using a FACS Canto II Flow cytometer and analyzed on FlowJo Software vX 10.0.7.
Facscanto 2 flow cytometer
Manufactured by FlowJo
Sourced in United States
The FACSCanto II flow cytometer is a versatile instrument designed for high-performance flow cytometry analysis. It features a compact design and offers a range of capabilities, including the ability to detect up to 10 parameters simultaneously. The FACSCanto II is capable of analyzing a variety of sample types, including cells, microparticles, and other biological particles.
Automatically generated - may contain errors
Lab products found in correlation
Cm h2dcfda, by Thermo Fisher Scientific (2 mentions)
Cd19 percp cy5, by BD (2 mentions)
Igd pe, by BD (2 mentions)
Cd20 percp cy5, by BD (2 mentions)
Cd38 pe cy7, by BD (2 mentions)
Ionomycin, by Merck Group (2 mentions)
Facscanto 2, by BD (2 mentions)
Golgiplug, by BD (2 mentions)
Perm wash buffer, by BD (2 mentions)
Cytofix cytoperm, by BD (2 mentions)
Carboxyfluorescein succinimidyl ester (cfse), by Thermo Fisher Scientific (2 mentions)
Cd138 apc, by BD (1 mentions)
Cd20 apc, by BD (1 mentions)
Cd19 alexa 700, by BD (1 mentions)
Iga pe, by BD (1 mentions)
Recombinant mouse ifn γ, by BioLegend (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Human ab serum, by Thermo Fisher Scientific (1 mentions)
Phorbol myristate acetate (pma), by Merck Group (1 mentions)
41 protocols using facscanto 2 flow cytometer
1
Quantifying Intracellular ROS Levels
2
Quantifying Intracellular ROS Levels
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The levels of intracellular ROS were measured using fluoresecent dye 2,7-dichlorofluorescein diacetate (DCF-DA). Cells were stained with 1 μM CM-H2DCFDA (Invitrogen Life Technologies, C6827) in Hank’s balanced salt solution for 30 min. Then cells were fixed with 4% paraformaldehyde before collecting cells. The stained cells were analyzed with FACSCanto II flow cytometer, and data were processed with the FlowJo software (FLOWJO).
3
Multiparametric Phenotyping of B Cells
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PBMCs were resuspended in phosphate-buffered saline (PBS), containing 0.5 % (w/v) BSA and 0.01 % sodium azide. PBMCs were incubated with saturating concentrations of fluorescently labeled conjugated monoclonal antibodies (MoAbs). Analysis of cells was performed using a FACSCanto-II flowcytometer and FlowJo software. The following directly conjugated MoAbs were used for flow cytometry: CD19-PerCP-Cy 5.5, CD19 Alexa-700, CD20-APC, CD20-PerCP-Cy 5.5, CD38 PE-Cy7, CD138 APC, IgG-PE, IgD-PE, and IgA-PE from BD-biosciences (San Jose, USA), CD27-FITC from Sanquin (Amsterdam, the Netherlands), IgM-PE from ITK-diagnostics (Uithoorn, the Netherlands).
4
Multiparameter Flow Cytometry Protocol
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Single cell suspensions were Fc-receptor-blocked for 30 min at 4°C with rat anti-mouse CD16/CD32 Ab in PBS with 1% FBS. Blocked cells were subsequently incubated with fluorophore-conjugated primary Abs for 60 min at 4°C, prior to washing in PBS containing 1% FBS and 2 mM EDTA. Following surface staining, cells were washed and analyzed by flow cytometry, or were fixed with 2% formaldehyde in PBS and then washed/permeabilized with BD perm/wash buffer (BD Biosciences), and stained with fluorophore-conjugated primary Abs for 60 min at 4°C. Cells were washed and intracellular fluorescence analyzed using a FACS Canto II flow cytometer and FlowJo software.
5
MHC Peptide Presentation and CD80 Analysis
6
RRMS Immune Profiling with Cladribine
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Cryopreserved PBMCs from 20 of the untreated patients with RRMS, 20 cladribine-treated patients at W52, 20 cladribine-treated patients at W96, and an additional 20 healthy controls were thawed and cultured for 48 hours in RPMI 1640 medium supplemented with penicillin and streptomycin (50 U/ml Gibco, Waltham, MA, USA) and 10% human AB serum (Invitrogen, Carlsbad, CA, USA). Cells were either left unstimulated or stimulated with CpG-ODN 2006 TLR9 agonist (InvivoGen, San Diego, CA, USA) at a concentration of 2.5 μg/ml. To assess B cell cytokine production, the CpG-stimulated and unstimulated PBMCs were subsequently incubated with phorbol-myristate-acetate (PMA; Sigma-Aldrich, St. Louis, MO, USA) at a concentration of 10 ng/ml, 0.5 μg/ml ionomycin (Sigma-Aldrich), and 5 μg/ml brefeldin A (Sigma-Aldrich), at 37°C and 5% CO2 for 4 hours. Following incubation, cells were surface stained with fluorochrome-conjugated anti-CD19 antibody (PE/Cy7; HIB19) and a live/dead stain. Subsequently, cells were fixed, permeabilized, and stained with anti-IL-10 (APC; JES3-19F1), anti-TGF-β (BV421; TW7-16B4) and anti-LTα (PE; 359-81-11) all from BioLegend. Flow cytometry data acquisition was performed using a FACS Canto II flow cytometer and analyzed using FlowJo software.
7
Assessing Murine Immune Cell Viability and Apoptosis
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Bone marrow cells and splenocytes were isolated from 8-week-old wild type (WT) and Hax1−/− mice, and red blood cells were lysed using 1x BD Pharm Lyse buffer (BD Biosciences). In total, 1 × 106 washed and pelletized cells were resuspended in 1 ml supplemented RPMI medium (described above), seeded in 48-well plates and incubated in a humidified atmosphere at 37°C.
Viability assay. The cells received no additional stimuli. Duplicates (1 × 106 per well) were analyzed for their viability from days 0 to 4. The cells were harvested, washed with 1 ml 1x PBS, and stained with eFluor780 for 30 minutes at 4°C. The stained cells were washed two times with 1x PBS.
Apoptosis assay. In total, 5 × 106 cells were stimulated with 2 μg goat anti-IgM (Southern Biotechnology, clone 1020-01). Then, 1 × 106 cells were analyzed at days 0, 1, 2, 3, and 4. The cells were washed with PBS and stained with eFluor450 and CD45R (B220) for 30 minutes at 4°C. After the cells were washed with 1 ml FACS buffer (1x PBS and 3% FCS) and 1 ml 1x binding buffer (eBioscience), they were stained with 5 μl annexin V-FITC (eBioscience), which was diluted in 100 μl 1x binding buffer, for 15 minutes in the dark at room temperature (RT). Then, the cells were washed with 1x binding buffer and apoptosis analysis was performed using a FACSCantoII flow cytometer and FlowJo 9.7.6 software.
Viability assay. The cells received no additional stimuli. Duplicates (1 × 106 per well) were analyzed for their viability from days 0 to 4. The cells were harvested, washed with 1 ml 1x PBS, and stained with eFluor780 for 30 minutes at 4°C. The stained cells were washed two times with 1x PBS.
Apoptosis assay. In total, 5 × 106 cells were stimulated with 2 μg goat anti-IgM (Southern Biotechnology, clone 1020-01). Then, 1 × 106 cells were analyzed at days 0, 1, 2, 3, and 4. The cells were washed with PBS and stained with eFluor450 and CD45R (B220) for 30 minutes at 4°C. After the cells were washed with 1 ml FACS buffer (1x PBS and 3% FCS) and 1 ml 1x binding buffer (eBioscience), they were stained with 5 μl annexin V-FITC (eBioscience), which was diluted in 100 μl 1x binding buffer, for 15 minutes in the dark at room temperature (RT). Then, the cells were washed with 1x binding buffer and apoptosis analysis was performed using a FACSCantoII flow cytometer and FlowJo 9.7.6 software.
8
Cytokine and Transcription Factor Analysis
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Cytokines, transcriptional factors and surface markers were evaluated by flow cytometry with a FACSCanto II (BD Biosciences). To detect intracellular expression of IL-17A, IFN-γ in CD4+ T cells, lymph nodes or CNS (purified with Percoll) were first treated with 750 ng ml−1 ionomycin (Sigma), 50 ng ml−1 phorbol 12-myristate 13-acetate (PMA) (Sigma) and GolgiPlug (BD Biosciences) for 4–6 h at 37 °C. Cells were fixed and permeabilized with the Foxp3 Staining Buffer Set (eBioscience) or BD Cytofix/Cytoperm (BD Biosciences) and were stained with fluorescent antibodies. After washing, stained cells were assayed with a BD Biosciences FACSCanto II flow cytometer and data were analysed with FlowJo software. For flow cytometry, monoclonal antibodies against CD4 (clone GK1.5), CD8 (clone 53-6.7), CD62L (clone MEL-14), CD44 (clone IM7), CD25 (clone PC61.5), IL-17A (clone eBio17B7), IFN-γ (clone XMG1.2), Helios (22F6, Biolegend) and FoxP3 (clone FJK-16s) were from eBioscience and CD3 (clone 145-2C11) was from Biolegend.
9
Cell-Mediated Cytotoxicity Assay with CFSE and 7-AAD
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K562 or Beas-2B cells were labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE) to assess cell-mediated cytotoxicity, using a 7AAD/CFSE Cell-mediated cytotoxicity assay kit (Ann Arbor, MI, USA). In total, 107 cells/mL were resuspended in CFSE staining solution and incubated for 15 min at 37 °C. Control target cells were resuspended in 0.1% BSA. Then, cells were washed two times with culture medium and incubated for 30 min at 37 °C. NK cells were put in co-culture with CFSE-labeled infected cells at a 1:5 ratio. The cell mixture was incubated for 4 h, centrifuged, and resuspended in 7-AAD staining solution. Control target cells were resuspended in assay buffer. Cells were incubated for 15 min in the dark at 4 °C. Then cells were centrifuged and resuspended in assay buffer. Cells were analyzed with a FACSCantoII flow cytometer and FlowJo software.
10
Selective PI3P Staining on Phagosomes
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For selective PI3P staining on the phagosomes, the 2×FYVE domain of HRS fused to green fluorescent protein (GFP–2×FYVEHrs) was utilised. For phagosome staining, RAW264.7 cells were pre‐treated for 2 h with 1 μM or 2 μM GSK2578215A and/or HG‐10‐102‐01 prior pulsing cells with 1‐μm latex beads for 30 min. After homogenisation in hypotonic buffer (3 mM imidazole, 250 mM sucrose and inhibitor of proteases and phosphatases), the post‐nuclear fraction containing phagosomes was fixed with 1% paraformaldehyde (PFA) in PBS for 10 min on ice. Fixation was stopped by the addition of PBS‐glycine at a final concentration of 0.2 M. The post‐nuclear supernatants were incubated for 30 min at RT with 5 μg/ml FYVE domain conjugate in PBS supplemented with 0.1% bovine serum albumin (BSA). Preparations were analysed after gating on a particular FSC/SSC region (corresponding to single beads in a solution). Samples were acquired on a BD FacsCanto II flow cytometer and analysed by FlowJo software v10.
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