PASMCs (2 × 105 cells) were cultured in 60 mm dish with 0.2% FBS DMEM for 24 h, and then switched to DMEM containing 10% FBS for 24 h. Harvested cells by mild trypsinization and centrifugation. Pre-chilled phosphate buffered solution (PBS) washed cells twice and then used pre-chilled 70% ethanol to fix cells at 4 °C overnight. The next day, cells were collected by centrifugation and washed with 1 mL of PBS, then added 500 μL PBS containing 50 μg/mL propidium iodine (PI), 100 μg/mL RNase A, 0.2% Triton X-100, incubating at 4 °C for 30 min in the dark. Cell cycle assay was determined by flow cytometry (CytoFLEX A00-1-1102; Beckman Coulter, Brea, CA, USA), and data were analyzed by using CytExpert software (Beckman Coulter). Used ModFit LT 3.2 software (Verity Software House, Topsham, ME, USA) to measure the percentage of cells in each phase of the cell cycle.
Cytoflex a00 1 1102
Manufactured by Beckman Coulter
Sourced in United States
The CytoFLEX A00-1-1102 is a flow cytometry instrument manufactured by Beckman Coulter. It is designed for the analysis of cells and particles in suspension.
Automatically generated - may contain errors
Lab products found in correlation
Cytexpert, by Beckman Coulter (2 mentions)
Modfit lt 3, by Verity Software House (1 mentions)
Cytexpert software, by Beckman Coulter (1 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
Ic fixation buffer, by Thermo Fisher Scientific (1 mentions)
Brefeldin a, by Thermo Fisher Scientific (1 mentions)
Permeabilization buffer, by Thermo Fisher Scientific (1 mentions)
Cytexpert 2, by Beckman Coulter (1 mentions)
Pe mouse igg1, by BD (1 mentions)
Fitc rat igg2a, by BD (1 mentions)
Facs tube, by BD (1 mentions)
Fitc conjugated cd14, by BD (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by ZSGB-BIO (1 mentions)
Annexin 5 fitc pi apoptosis detection kit, by Beyotime (1 mentions)
Trypsin, by Thermo Fisher Scientific (1 mentions)
2 4 amidinophenyl 6 indolecarbamidine dihydrochloride dapi, by Beyotime (1 mentions)
8 protocols using cytoflex a00 1 1102
1
Cell Cycle Analysis by Flow Cytometry
2
ROS Quantification in Cells
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Levels of ROS within cells were assessed using a ROS kit (cat. no. S0033S; Beyotime Institute of Biotechnology). First, cells were digested to obtain a cell suspension. Then, the cell suspension was incubated in a medium without serum, ultimately reaching a concentration of 40 mM for 20 min at 37˚C. The staining lasted for 1 h at 15˚C. A flow cytometer (CytoFLEX A00-1-1102; Beckman Coulter, Inc.) and its corresponding software CytExpert (Version 2.4; Beckman Coulter, Inc.) were used to detect the ROS fluorescence intensity.
3
Cytokine Expression in DC-T Cell Interactions
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In vitro DC-T cell co-cultures were incubated for 8 h. Cells were then harvested and stained with a CD69 antibody (eBioscience), and CD69 expression was analyzed using flow cytometry. The co-cultures were incubated for 72 h, and 3 μg/ml Brefeldin A (eBioscience) was added for the last 5 h. Then, the cells were harvested, washed in RPMI-1640 and fixed with IC fixation buffer (eBioscience) for 30 min. Afterward, the cells were permeabilized in 1× permeabilization buffer (eBioscience) and incubated with fluorochrome tagged anti-IFN-γ-phycoerythrin (APC; eBioscience) and anti-IL-17A-fluorescein isothiocyanate (PE; eBioscience) for 90 min. The labeled cells were detected using flow cytometry (CytoFLEX A00-1-1102; Beckman Coulter, USA). Standard washing and incubation protocols were followed at each stage.
4
Cell Cycle Analysis by Flow Cytometry
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Cells (5x106) were collected and fixed in 70% ice-cold ethanol at -20˚C overnight. The cells were then collected and resuspended in PBS supplemented with 100 ng/ml RNase A and 50 ng/ml propidium iodide (PI) for 30 min at 37˚C. After staining, the distribution of cell cycle stage was assessed using a flow cytometer (CytoFLEX A00-1-1102, Beckman Coulter Biotechnology) and analysis software (CytExpert 2.4, Beckman Coulter Biotechnology).
5
Flow Cytometric Analysis of CD14 and TLR4 Expression
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The cells in each group were collected into FACS tube (352063, BD) and washed in ice-cold phosphate-buffered saline (PBS) twice. THP-1 and PMA-differentiated THP-1 cells were stained with fluorescein isothiocyanate (FITC)-conjugated CD14 (555397, BD Biosciences, Franklin Lakes, NJ, USA) and phycoerythrin (PE)-conjugated TLR4 (564215, BD Biosciences) for 1 h at room temperature. The stained cells were analyzed by flow cytometry (cytoFLEX, A00-1-1102, Beckman Coulter, Brea, CA, USA) and software (CytExpert, Beckman Coulter). For each sample, the count in 10,000 cells was measured. The region of each sample was selected in the forward scatter and side scatter, and then a histogram was used to measure the mean fluorescence intensity of the FITC or PE, which represented the CD14 or TLR4, respectively. FITC-rat IgG2a (BD, 555843) and PE-mouse IgG1 (BD, 559320) were used as isotype controls.
6
Apoptosis Analysis in Cardiomyocytes
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The apoptosis was evaluated by the terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) staining method, using a kit from Beyotime Institute of Biotechnology, China. Nuclei were stained with DAPI (ZSGB-BIO). In each experiment, cells in at least three random microscopic fields were imaged for the analysis.
Besides, assessment of cardiomyocyte apoptosis using Annexin V-FITC and PI. Cells were seeded at 106 cells per well in a 6-well plate and treated after 24 hours of drug treatment. The assessment of the apoptosis of cardiomyocytes was performed by using an Annexin V-FITC/PI. The assessment of the apoptosis of the cardiomyocytes was performed by using an Annexin V-FITC/PI Apoptosis Detection Kit (Beyotime Institute of Biotechnology, Beijing, China). The stained cells were analyzed by flow cytometry (CytoFLEX A00-1-1102, Beckman, USA) to evaluate cellular damage.
Besides, assessment of cardiomyocyte apoptosis using Annexin V-FITC and PI. Cells were seeded at 106 cells per well in a 6-well plate and treated after 24 hours of drug treatment. The assessment of the apoptosis of cardiomyocytes was performed by using an Annexin V-FITC/PI. The assessment of the apoptosis of the cardiomyocytes was performed by using an Annexin V-FITC/PI Apoptosis Detection Kit (Beyotime Institute of Biotechnology, Beijing, China). The stained cells were analyzed by flow cytometry (CytoFLEX A00-1-1102, Beckman, USA) to evaluate cellular damage.
7
One-Step Growth Curve of Bacteriophages
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A one-step growth curve was determined to measure the incubation period. Briefly, 500 μL of purified phage solution and 500 μL of bacteria solution in the logarithmic growth phase were mixed (MOI=0.1), and adsorption at 30°C for 10 min. The mixture was then centrifuged at 8,000g for 2 min, and the precipitate was resuspended with 2216E liquid medium, then centrifuge was repeated 3 times to remove the remaining unabsorbed phages, and then sampling was repeated every 10 min. The experiment was conducted in triplicate (Cai et al., 2019 (link)). The number of viral particles was calculated by flow cytometry (Beckman Coulter CytoFLEX, A00-1-1102).
8
Cellular Uptake of QCDA in HepG2 Cells
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The HepG2 cells were seeded at a density of 1 × 105 cells into CLSM dishes and 6-well plates for 24 h. Then, the media were removed and the fresh DMEM containing QCDA (16 μg/mL) was added to the dishes for 2, 4 and 8 h. For the dishes, the media were sucked out, and the dishes were rinsed gently with phosphate-buffered saline (PBS) three times following fixation in 4% paraformaldehyde (PFA; Shanghai Macklin Biochemical Technology Co., Ltd., Shanghai, China) for 15 min and staining with 100 μL of 2-(4-Amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI; Beyotime, Shanghai, China) for 25 min at 37 °C. After being washed three times with PBS, the cellular uptake of QCDA was observed by a confocal laser scanning fluorescence microscope (CLSM; NIKON A1, Nikon Corporation, Tokyo, Japan). The 6-well plates were washed with PBS three times after removing the media. Then, 300 μL of trypsin (Gibco, Beijing, China) was added into the well for the collection of cells. After being washed three times with PBS, the cellular uptake of QCDA was observed via flow cytometry (Cytoflex A00-1-1102, Beckman Coulter, Suzhou, China).
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