Streptavidin has previously been shown to bind with high specificity to 8-oxoG [49 (link),50 (link)] and was therefore used for the 8-oxoG measurements. Epimastigotes and amastigotes parasites were treated with H2O2 for 30 minutes, fixed in paraformaldehyde (2% v/v in PBS) at 25°C for 15 min and thereafter incubated for 15 min in PBS with 0.1% Triton X-100 v/v. Cells were then incubated with Alexa488-conjugated streptavidin (Invitrogen) (1:100) in PBS for 1 h at 37°C and evaluated by flow cytometer (FACS-Calibur).
Facscalibur
Manufactured by Thermo Fisher Scientific
Sourced in United States, Germany
The FACSCalibur is a flow cytometry system designed for cell analysis and sorting. It utilizes dual-laser technology to detect and measure multiple parameters of individual cells or particles within a fluid sample. The FACSCalibur is capable of analyzing a wide range of cell types and providing quantitative data on their physical and fluorescent characteristics.
Automatically generated - may contain errors
Lab products found in correlation
Facscalibur, by BD (6 mentions)
Cytofix cytoperm buffer, by Thermo Fisher Scientific (3 mentions)
Brefeldin a, by Merck Group (3 mentions)
Ionomycin, by Merck Group (3 mentions)
Annexin 5 apoptosis detection kit apc, by Thermo Fisher Scientific (2 mentions)
Microwin software, by Berthold Technologies (2 mentions)
Flowcytomix, by Thermo Fisher Scientific (2 mentions)
Flowcytomix pro software, by Thermo Fisher Scientific (2 mentions)
Cellquest software, by BD (2 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (2 mentions)
Alexa 488 conjugated streptavidin, by Thermo Fisher Scientific (1 mentions)
Mitosox red superoxide indicator, by Thermo Fisher Scientific (1 mentions)
Cm h2dcfda, by Thermo Fisher Scientific (1 mentions)
Mitosox red, by Thermo Fisher Scientific (1 mentions)
Facscalibur flow cytometer, by BD (1 mentions)
Live dead fixable far red dead cell staining kit, by Thermo Fisher Scientific (1 mentions)
Propidium iodide, by BD (1 mentions)
Odn2006, by InvivoGen (1 mentions)
αigm f ab 2, by Jackson ImmunoResearch (1 mentions)
Aim 5 medium, by Thermo Fisher Scientific (1 mentions)
43 protocols using facscalibur
1
Quantifying Oxidative Stress in Parasites
2
Mitochondrial Oxidative Stress Assay
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Cells were incubated with 2 uM CM-H2DCFDA (Molecular Probes/Invitrogen) resuspended in warm HBSS or HBSS alone for unstained controls for exactly 15 min. The cells were analyzed on a FACSCalibur flow cytometer (BD). The cellular subset was identified according to size and granularity. We used a mitochondria-specific dye (MitoTracker Green FM) that binds the mitochondrial membrane independently of the membrane potential, and thus, the staining intensity is considered an index of mitochondrial mass. For MitoSOX Red-based flow cytometric detection of mitochondrial superoxide, cells were then incubated with MitoSOX Red superoxide indicator (Invitrogen) for 30 min, washed, and then analyzed on a FACSCalibur. The mean channel fluorescence was converted to absolute fluorescence using an inverse log transformation and normalized to that of untreated cells or WT hepatocytes.
3
Quantitative Cell Death Assay
4
Annexin V-PI Apoptosis Assay
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Apoptotic cell death was analysed by Annexin V and Propidium Iodide double staining following 24/48 h of esculetin treatment according to the manufacturer’s instructions (Invitrogen) using FACSCalibur and Flowing software.
5
Stimulation of B cells with ODN2006 and αIgM
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B cells were isolated from human frozen PBMCs using EasySep Human CD19 Positive Isolation Kit (StemCell Technologies) according to manufacturers’ protocol. The cells were seeded in AIM V medium (Gibco). The cells were pre-treated for 1 h with different concentrations of CPL302-253 and subsequently stimulated 1μg/ml ODN2006 (Invivogen) and 10μg/ml αIgM F(ab’)2(Jackson ImmunoResearch). After 72 hours of culture the cells were stained with antibody againstCD25 (BC96, eBioscience) and ki-67 (Clone 20Raj1, Invitrogen) and analysed on FACSCalibur.
6
In vitro T cell stimulation
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The in vivo primed T cells were stimulated for 5 days in vitro with the immunizing antigen and APCs, then activated T cells were separated using Ficoll gradient centrifugation and stimulated in vitro for 4 h with 50 ng/ml of phorbol myristic acetate (PMA), 1 μg/ml of ionomycin, and 1 μg/ml of brefeldin A (Sigma, St. Louis, MO). The cells were then fixed, permeabilized overnight with Cytofix/Cytoperm buffer (eBioscience, San Diego, CA), intracellularly stained with Abs against IFN-γ or IL-17, and analyzed on a FACScalibur.
7
Cell Cycle and Apoptosis Analysis
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AGS and MGC-803 cells of each group (shRPL31 and shCtrl) were seeded in the well dishes (6 cm) and cultured until cell coverage rate was about 80%. After that, cells were washed by PBS, processed by trypsin and centrifuged for 5 min (1200 rpm). To detect cell cycles, pre-cooled ethanol (70%) was used to fix cell suspension and then removed at least 1 h later. After centrifuged and washed by PBS, cells were stained with PI staining solution (40 × PI, 2 mg/mL: 100 × RNase, 10 mg/mL: 1 × PBS = 25:10:1000) for half an hour. Then we adopted FACSCalibur (eBioscience, San Diego, USA) to detect cell cycle. As to the assessment of cell apoptosis, we stained cells using Annexin V-APC for 15 min in dark and determined the percentage of cells in each mitotic phase to calculate cell apoptotic rate.
8
Purification and Activation of CD4+ T Cells
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CD4+ T lymphocytes were purified from PBMCs isolated from three anonymous blood donors (with ages ranging from 18 to 65 years) by negative selection using the Human CD4+ T Cell Enrichment Kit (#19052, Stemcell Technologies) according to the manufacturers' instructions. Purification was over 90% as assessed by staining with 5 ng/μl of anti-CD4-FITC (clone RPA-T4, eBioscience) and flow cytometric analysis (FACSCalibur).
Isolated CD4+ T lymphocytes (2 × 105) were resuspended in RPMI medium (control) or preincubated for 24 hours with 50% of AMSC-derived CM and SF, then seeded in triplicate into 96-well plates with prebound 0.5 μg/ml anti-CD3 (clone OKT3, eBiosciences), 0.5 μg/ml anti-CD28 (clone CD28.6, eBiosciences), and recombinant IL-2 at a concentration of 250 U/ml (Peprotech). After 3 days, in vitro-stimulated CD4+ T cells were stained with 5 ng/μl of anti-CD25-APC (clone BC96, eBiosciences) and 5 ng/μl of anti-FoxP3-PE (clone PCH101, eBioscience) and T reg proliferation was tested with flow cytometry (FACSCalibur, Becton Dickinson). The data were analysed using the Flowing Software.
Isolated CD4+ T lymphocytes (2 × 105) were resuspended in RPMI medium (control) or preincubated for 24 hours with 50% of AMSC-derived CM and SF, then seeded in triplicate into 96-well plates with prebound 0.5 μg/ml anti-CD3 (clone OKT3, eBiosciences), 0.5 μg/ml anti-CD28 (clone CD28.6, eBiosciences), and recombinant IL-2 at a concentration of 250 U/ml (Peprotech). After 3 days, in vitro-stimulated CD4+ T cells were stained with 5 ng/μl of anti-CD25-APC (clone BC96, eBiosciences) and 5 ng/μl of anti-FoxP3-PE (clone PCH101, eBioscience) and T reg proliferation was tested with flow cytometry (FACSCalibur, Becton Dickinson). The data were analysed using the Flowing Software.
9
Apoptosis Quantification by FACS
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Cells were collected and detected by routine DAPI (4′,6‐diamidino‐2‐phenylindole) staining using a commercial DAPI dye (Beyotime, Shanghai, China) and Annexin V Apoptosis Detection Kit APC (eBioscience, San Diego, CA, USA) according to the manufacturers’ instructions and analysed by FACS Calibur. Briefly, 1 × 106 cells were washed and resuspended in 1× binding buffer. Fluorochrome‐conjugated Annexin V was added to the cell suspension and incubated at room temperature for 10 minutes. Cells were washed with 1× binding buffer. Propidium iodide (50 μg/mL) was added before flow cytometry analysis.
10
Measuring T Cell Activation via miR-27
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