Macrophages grown in 6-well tissue culture plates were infected with FMDV as described earlier and cells were harvested at 0, 4, 12 and 18 hpi. After treatment, cells were washed with PBS and detached from culture plates by adding PBS containing EDTA (5 mmol/L) and incubated at 4 °C for 10 min, before thorough pipetting. 1 × 106 cells per sample were used for analysis by Cytomics FC500 flow cytometer (Beckman Coulter, USA) and stained with the following antibodies: APC-conjugated anti-mouse F4/80 antibody, PE-conjugated anti-mouse CD11c and FITC-conjugated anti-mouse CD206 (all antibodies were from BioLegend, USA). Antibodies were diluted in FACS buffer (PBS containing 5 % FBS, pH 7.4). Each antibody was incubated at 4 °C for 15 min in the dark. Cells were washed twice with FACS buffer between each antibody incubation. After washing, cells were resuspended in FACS buffer and run on a Cytomics FC500 flow cytometer (Beckman Coulter, USA). M1 macrophages were identified as F4/80-positive/CD11c-positive while M2 macrophages were identified as F4/80-positive/CD206-positive. The data was analyzed by CXP Software Version 2.2 (Beckman Coulter, USA).
Cytomics fc500 flow cytometer
Manufactured by Beckman Coulter
Sourced in United States, Germany, China, Canada, France, Ireland
The Cytomics FC500 is a flow cytometer designed for research applications. It utilizes laser technology to analyze and sort cells or particles in a fluid stream. The device can measure multiple parameters, including size, granularity, and fluorescence, to characterize and classify different cell types or populations.
Automatically generated - may contain errors
Lab products found in correlation
Cxp software, by Beckman Coulter (23 mentions)
Cxp analysis software, by Beckman Coulter (19 mentions)
Cxp software version 2, by Beckman Coulter (6 mentions)
Rnase a, by Merck Group (5 mentions)
Streptavidin energy coupled dye, by Beckman Coulter (4 mentions)
Kaluza software, by Beckman Coulter (4 mentions)
Pi rnase staining buffer, by BD (4 mentions)
Cytofix cytoperm, by BD (4 mentions)
Flow count fluorosphere, by Beckman Coulter (4 mentions)
Trypsin edta, by Thermo Fisher Scientific (4 mentions)
Immunoprep reagent system, by Beckman Coulter (3 mentions)
Mouse fcr blocking reagent, by Miltenyi Biotec (3 mentions)
Modfit lt, by Verity Software House (3 mentions)
Propidium iodide solution, by Cytognos (3 mentions)
Multicycle av software, by Phoenix Pharmaceuticals (3 mentions)
Kaluza software version v2, by Beckman Coulter (3 mentions)
Cell cycle phase determination kit, by Cayman Chemical (3 mentions)
Propidium iodide, by Merck Group (3 mentions)
Rnase a, by Thermo Fisher Scientific (3 mentions)
Propidium iodide, by BD (3 mentions)
357 protocols using cytomics fc500 flow cytometer
1
Macrophage Polarization in FMDV Infection
2
Immune and Inflammatory Profiles in COVID-19
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Throat-swab specimens from the upper respiratory tract that were obtained at admission. The novel coronavirus nucleic acid was confirmed by real-time RT-PCR (cobas® z 480 Automatic Fluorescence Quantitative PCR, Roche, USA) with a commercial kit (Pfizer, USA). FBG was determined by the glucose oxidase method (Abbott original reagent, by Architect c16000, Abbott, USA), and HbA1c was tested by high-performance liquid chromatography (Bio-Rad original reagent, by Bio-Rad VARIANT II, Bio-Rad, USA). To identify the immune and inflammatory characteristics, peripheral blood samples were collected for detection of T cell subsets (CD4+, CD8+) (Beckman Coulter original reagent, by Cytomics FC 500 Flow Cytometer, Beckman Coulter, USA) and cytokines (IL6, TNFα, IL2, IL4, IL10, and IFNγ), using flow cytometry (commercial kits of BD, USA, by Cytomics FC 500 Flow Cytometer, Beckman Coulter, USA). The peripheral blood of patients was sampled before they used Traditional prescription [17 (link), 18 (link)] (“Xinguan No.1”, “Xinguan No.2”, or “Xinguan No.3”), tocilizumab [19 (link)] and glucocorticoids [20 (link)], considering their effects on inflammation or immune regulation. All indices were tested immediately or on the next morning after admission.
3
Cell Cycle Analysis by Flow Cytometry
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siRNA-transfected cells were collected, washed twice with PBS and fixed in ice-cold 70% ethanol overnight at −20 ºC. Then, cells were treated with 100 μg/ml RNase A in PBS, and incubated at 37 °C for 30 min before staining with 50 μg/ml of PI (BD Biosciences) for 30 min at room temperature. Cells were analyzed for DNA content using a Cytomics FC-500 flow cytometer (Beckman Coulter).
For retroviral transfected cells, cells were first fixed with 1% formaldehyde for 1 h at 4 ºC, washed once with PBS and permeabilized with ice-cold 70% ethanol overnight at −20 ºC. Then, cells were treated with 100 μg/ml RNase A in PBS, and incubated at 37 °C for 30 min before staining with 50 μg/ml of PI for 30 min at room temperature. GFP-positive cells were analyzed for DNA content using a Cytomics FC-500 flow cytometer (Beckman Coulter).
For retroviral transfected cells, cells were first fixed with 1% formaldehyde for 1 h at 4 ºC, washed once with PBS and permeabilized with ice-cold 70% ethanol overnight at −20 ºC. Then, cells were treated with 100 μg/ml RNase A in PBS, and incubated at 37 °C for 30 min before staining with 50 μg/ml of PI for 30 min at room temperature. GFP-positive cells were analyzed for DNA content using a Cytomics FC-500 flow cytometer (Beckman Coulter).
4
Multicolor Flow Cytometry for Apoptosis and Cell Phenotyping
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Double staining of annexin V and PI was performed using Annexin V Apoptosis detection set phycoerythrin (PE)-Cy7 (eBioscience, San Diego, CA, USA) or Brilliant Violet 421™ (BV421) Annexin V (BioLegend, San Diego, CA, USA), and activated caspases detection was performed using a FLICA 660 caspase-1 or caspase-3/-7 assay kit (Immunochemistry Technologies, Bloomington, MN, USA) according to the manufacturer's protocol. Stained cells were analyzed with a flow cytometer Cytomics FC 500 (Beckman Coulter, Fullerton, CA, USA). Triple staining of CD11b, Ly6C, and Ly6G for 32D/TetOff-p210 cells was performed using anti-CD11b-BV421, anti-Ly6C-allophycocyanin (APC) (BD Biosciences), and anti-Ly6G-PE (BD Biosciences), respectively. Stained cells were analyzed with a high-speed cell sorter MoFlo AstriosEQ (Beckman Coulter, Fullerton, CA, USA). Triple staining of CD11b, Ly6C, and Ly6G for spleen cells from mice was performed using anti-CD11b-PE (BD Biosciences) anti-Ly6C-APC (BD Biosciences), and anti-Ly6G-fluorescein isothiocyanate (FITC) (BD Biosciences), respectively. Stained cells were analyzed with a flow cytometer Cytomics FC 500 (Beckman Coulter).
5
Phenotyping Autologous CIK Cells
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The phenotype of the autologous CIK cells was characterized by flow cytometry. CIK cells were resuspended at 2 × 105 cells per 100 μL of phosphate-buffered saline (PBS) and incubated for 30 min at 4°C with the following anti-human antibodies: anti-CD3-PE-Cy5, anti-CD4-FITC, anti-CD8-PE-CF594 and anti-CD56-PE-Cy7 (all from BD Bioscicence). The cells were analyzed using a CytomicsTM FC500 Flow Cytometer (Beckman Coulter, USA). Data analysis was performed with CXP analysis software (Beckman Coulter, USA).
6
Annexin V-FITC Apoptosis Assay
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We used Annexin V/FITC to detect apoptotic cells. After UA treatment with Huh-7 cells, cells were collected and then rinsed with PBS. Cells were re-suspended in 100 μL binding buffer, and then 5 μL of Annexin V-FITC and 10 μL of PI were added for 15 min at RT in the dark. Thereafter, 500 μL of binding buffer was added. Then the samples were examined using a CytomicsTM FC500 flow cytometer (Beckman Coulter, FL, USA).
7
Bruceine D Mitochondrial Potential
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The ΔΨm of cells exposed to 1, 2.5 and 5 µg/ml bruceine D or vehicle alone was measured using the fluorescent cationic dye Rhodamine 123, according to the manufacturer's protocol (Molecular Probes; Thermo Fisher Scientific, Inc.). Cells were analyzed using a CytomicsTM FC500 flow cytometer (Beckman Coulter, Inc.).
8
Cell cycle analysis by flow cytometry
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The cells were washed by PBS two times, trypsinized, fixed with ice-cold 100% ethanol and kept on −20 °C for overnight. Cells were rehydrated with cold PBS, and then resuspended in PBS with propium iodine (40 µg/mL) (#P4170; Sigma-Aldrich, St. Louis, MO, USA) and Ribonuclease A (0.2 µg/mL) at room temperature for 30 min in the dark. The content of DNA in each sample were analyzed by a CytomicsTM FC500 flow cytometer (Beckman Coulter; Brea, CA, USA).
9
Cell Cycle and Apoptosis Analysis
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Cells were seeded in 6-well plates, incubated for 24 h, and treated with PBS, 5-azaC (1 μmol/l), IR (6 Gy), or 5-azaC + IR as described above. Cell cycle progression and apoptosis were analyzed with the Cell Cycle and Apoptosis Kit (Keygentec, China) using a CytomicsTM FC500 flow cytometer and CXP analysis software (Beckman Coulter, USA) following the manufacturer’s instructions. Cell cycle analysis was performed using CXP analysis software; apoptotic cells were considered to include cells stained Annexin V (+)/propidium iodide (PI) (−) (lower right quadrant, early apoptosis) and late cells stained Annexin V (+)/PI (+) (upper right quadrant, late apoptosis) [22] (link).For each sample, at least 10,000 cells were analyzed.
10
Cell Cycle Analysis by Flow Cytometry
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Following incubation for 48 h, cells were harvested and fixed overnight in cold 75% ethanol at 4°C. Next, cells were washed again with pre-cooled PBS and resuspended in 400 µl FxCycle™ PI/RNase staining solution (Molecular Probes; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. The samples were incubated for 30 min at room temperature in the dark, and then analyzed using a CytomicsTM FC500 flow cytometer (Beckman Coulter, Inc.). Cell cycle distribution was calculated with ModFit LT 4.1 software (Becton Dickinson and Company).
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