The cell number and viability were assessed using the ApoFlowEx FITC Kit (Exbio, Vestec, Czech Republic) each time before seeding the cells into culture dishes and at each of the four time points. For this purpose, pooled adherent and non-adherent hAECs were counted using a Cytoflex cytometer, and then batches of 200,000–500,000 cells were suspended in 100 µL of pre-diluted buffer (Annexin V Binding Buffer 10×). The cell suspension was supplemented with 5 µL Annexin V-FITC and 5 µL propidium iodine and incubated for 15 min at room temperature, followed by analysis on a Cytoflex cytometer.
Cytoflex cytometer
Manufactured by Beckman Coulter
Sourced in United States, France, Germany, Canada
The CytoFLEX is a flow cytometer designed for high-performance cell analysis. It features advanced optical and fluidic systems to deliver reliable and accurate data. The CytoFLEX is capable of detecting multiple fluorescent parameters simultaneously, enabling comprehensive analysis of cell populations.
Automatically generated - may contain errors
Lab products found in correlation
Cytexpert software, by Beckman Coulter (12 mentions)
Golgiplug, by BD (6 mentions)
Cytexpert acquisition and analysis software, by Beckman Coulter (6 mentions)
Cytofix cytoperm solution, by BD (6 mentions)
Live dead sperm viability kit, by Thermo Fisher Scientific (4 mentions)
Cd28 cd49d, by BD (4 mentions)
Foxp3 transcription factor staining buffer set, by Thermo Fisher Scientific (4 mentions)
Cytexpert 2, by Beckman Coulter (4 mentions)
Fluorophore conjugated antibodies, by Miltenyi Biotec (3 mentions)
Ifn γ and tnf α antibodies, by Miltenyi Biotec (3 mentions)
Golgistop, by BD (3 mentions)
Fix perm kit, by Thermo Fisher Scientific (3 mentions)
Zombie red fixable viability kit, by BioLegend (3 mentions)
Kaluza 2, by Beckman Coulter (3 mentions)
Propidium iodide, by Merck Group (3 mentions)
Mitotracker red, by Thermo Fisher Scientific (3 mentions)
Annexin 5 pe 7 aad apoptosis detection kit, by Keygen Biotech (2 mentions)
Cd56 bv650, by Miltenyi Biotec (2 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (2 mentions)
Fix and perm kit, by Thermo Fisher Scientific (2 mentions)
182 protocols using cytoflex cytometer
1
Assessing Cell Viability and Apoptosis
2
Apoptosis and Cell Cycle Analysis
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Apoptosis was assessed using the Annexin V-PE/7-AAD Apoptosis Detection Kit (KeyGen Biotech, Nanjing, China) according to the manufacturer’s instructions. Briefly, cells that had been harvested with trypsin, without EDTA, were washed twice with cold PBS and then resuspended in Binding Buffer. Fifty microliters of the cell suspension (100,000 cells) was then transferred to a flow cytometry tube, and 5 μL of 7-aminoactinomycin was added. After incubation for 5-15 min in the dark, 450 μL of Binding Buffer and 1 μL of annexin V-phycoerythrin were added to each tube. After incubation for 5-15 min in the dark, flow cytometric analysis was performed using a CytoFLEX Cytometer (Beckman-Coulter, Brea, CA, USA).
Each phase of the cell cycle was analyzed by flow cytometry using a Cell Cycle Detection Kit (KeyGen Biotech). Briefly, cells were collected and fixed with 75% ethanol overnight at 4°C. The cells were then washed in PBS and stained with propidium iodide/RNase staining buffer for 30-60 min at room temperature in the dark. Flow cytometric analysis was performed using a CytoFLEX Cytometer (Beckman-Coulter).
Each phase of the cell cycle was analyzed by flow cytometry using a Cell Cycle Detection Kit (KeyGen Biotech). Briefly, cells were collected and fixed with 75% ethanol overnight at 4°C. The cells were then washed in PBS and stained with propidium iodide/RNase staining buffer for 30-60 min at room temperature in the dark. Flow cytometric analysis was performed using a CytoFLEX Cytometer (Beckman-Coulter).
3
Cell Cycle and Apoptosis Analysis
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Tumor cells were plated in six-well plates at 3 × 105 cells per well. After 24 h incubation, cells in each group were treated for 24 h to analyze the cell cycle and for 48 h to measure cell apoptosis. Cells incubated in medium without treatment were used as controls. For cell cycle analysis [35 (link)], the cells were harvested with trypsin, washed with cold PBS and fixed with 70% ethanol at 4 °C overnight. Before analysis, the cells were washed with PBS and stained with 50 μg/mL PI and 50 μg/mL RNase at 37 °C for 15 min. Finally, samples were immediately measured with a CytoFLEX cytometer (Beckman Coulter, Pasadena, CA, USA). For cell apoptotic measurements [36 (link)], the cells in different treated groups were harvested, washed with PBS and then treated with trypsin-EDTA solution to detach the cells. The suspended cells were centrifuged at 200× g for 10 min. After being washed twice with cold PBS, cells were collected by centrifugation at 200× g for 5 min and stained with FITC-conjugated Annexin V and PI. Then, the samples were immediately measured with a CytoFLEX cytometer (Beckman Coulter, Pasadena CA, USA).
4
Mouse T Cell Absolute Counts from Mandibular Vein
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T cell absolute count was performed on blood samples kept from the mandibular vein of the mouse.
For the phenotypic characterization of cell populations, the following antibodies were used: CD8-FITC (Miltenyi Biotec), CD25-APC (Pharmingen), CD3-PE-Vio770 (Miltenyi Biotec), CD4-APC-Vio770 (Milteny Biotec), CD45R (B220)-Violblu (Milteny Biotec), NK1.1-PE (Milteny Biotec).
At predetermined optimal concentrations, 100 μl of blood was stained by incubation with the antibodies. Fifty microliters of CountBright Absolute Counting Beads (Molecular Probes) was added, and, following lysis of red blood cells, cells were acquired on a CyAn Cytometer (Beckman Coulter). By comparing the ratio of bead events to cell events, absolute numbers of cells in the sample were calculated.
Some experiments were performed by acquiring the stained blood samples on the CytoFLEX cytometer (Coulter), equipped with a volumetric sample injection module, which enables volumetric sampling and provides absolute cell counts for all samples without the use of beads.
For the phenotypic characterization of cell populations, the following antibodies were used: CD8-FITC (Miltenyi Biotec), CD25-APC (Pharmingen), CD3-PE-Vio770 (Miltenyi Biotec), CD4-APC-Vio770 (Milteny Biotec), CD45R (B220)-Violblu (Milteny Biotec), NK1.1-PE (Milteny Biotec).
At predetermined optimal concentrations, 100 μl of blood was stained by incubation with the antibodies. Fifty microliters of CountBright Absolute Counting Beads (Molecular Probes) was added, and, following lysis of red blood cells, cells were acquired on a CyAn Cytometer (Beckman Coulter). By comparing the ratio of bead events to cell events, absolute numbers of cells in the sample were calculated.
Some experiments were performed by acquiring the stained blood samples on the CytoFLEX cytometer (Coulter), equipped with a volumetric sample injection module, which enables volumetric sampling and provides absolute cell counts for all samples without the use of beads.
5
Multiparametric Flow Cytometry of PBMCs
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PBMC were harvested after 6 hours of culture and resuspended in an EDTA-containing medium, then stained for 15 minutes at 4 degrees with the following antibodies: Anti-human CD45-FITC (Coulter; 1/200), OX40-L-PE (BD; 1/50), CD4-ECD (Coulter; 1/100), CD16-BB700 (Pharmigen; 1/100), PDL-1-PC7 (Coulter; 1/100), CD86-APC (Miltenyi; 1/80), CD11c-APC-AlexaFluor700 (Coulter; 1/100), CD14-APC-Vio770 (Miltenyi; 1/120), CD3-e450 (eBioscience; 1/160), CD19-PB (Dako; Santa Clara, CA, USA 1/60), CD56-BV421 (BD; 1/50), Fixable Aqua Dead Cell stain (Thermofisher; 1/200), CD123-BV605 (Biolegend, San Diego, CA, USA; 1/30), CD80-BV650 (BD; 1/50), and HLA-DR-BV785 (BD; 1/100). Samples were washed in EDTA-containing medium, acquired using a Cytoflex cytometer (Coulter) and analyzed using FlowJo-10 software (version 10.3.0).
6
Flow Cytometry for Microbial Abundance
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Flow cytometry was used to determine the abundance of autotrophic cells (66 (link)) and also the abundance of Sybr green-I-stained bacteria and heterotrophic nanoflagellates (67 (link)– 69 (link)). For the flow cytometry count of autotrophic cells, 2 ml of preserved samples in 0.5% glutaraldehyde was frozen at −80°C and stored until analysis (5 to 10 days). Cyanobacterial cells were distinguished according to light scattering, red emission of cellular chlorophyll content, and orange emission of phycoerythrin-rich cells. The autotrophic community was processed on a Cytoflex cytometer. Samples used for the analysis of bacterial abundances and heterotrophic nanoflagellates were preserved in 2% formaldehyde and stored at 4°C until analysis (5 to 10 days). The heterotrophic samples were processed on a Beckman Coulter EPICS XL-MCL instrument with a high flow rate from 1 to 1.2 μl s−1, and the analyzed volume was calculated according to the acquisition time.
7
Phagocytosis Assay with IFN-α Priming
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HD11 WT cells were seeded at a confluence of 3x10 5 cells/ml in 12-well plates. The cells were primed with IFNα for 16h and then with transfected exogenous DNA (HT-and CT-DNA -2 μg/mL) or treated with 2'3'cGAMP (5 μg/mL) for 6 h. After this, the cells were incubated with Zymosan coated beads conjugated with FITC at a ratio of 30 beads to 1 cell for all conditions for 40 min at 37 °C. The cells were wash two times in PBS and fixed in suspension using the solution (missing ref; BD Biosciences) with 4% PFA. Cell populations were counted by analysis on a CytoFLEX cytometer.
8
Quantifying Cellular Uptake of AuNPs
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The internalization (uptake) of AuNPs was determined using the method described by Park et al.13 (link) Cells were seeded on the 12-well plates at a density of 200,000 cells per well. After 24 h, RGD-PEG800-AuNPs10nm, RGD-PEG2000-AuNPs10nm, RGD-PEG800-AuNPs30nm, RGD-PEG2000-AuNPs30nm were added at 0.0004, 0.0008, 0.0012 mg/mL for each AuNP type. The level of AuNP internalization in cells was determined after 0.5, 1, 3, 6, and 24 h. Cells were harvested after incubation, suspended in Phosphate Buffered Saline (PBS) (Biowest, France), and washed once to discard excess AuNPs from the sample. Cytoflex Beckman Coulter cytometer (Beckman Coulter Life Sciences, Indianapolis, IN, USA) was used for cytometric analysis. Fluorescence was measured at 611 nm by analyzing the side scatter parameter (SSC). Analysis of the obtained results was performed using FlowJo v10.
9
Apoptosis Evaluation of Irradiated Cells
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Cells were irradiated and plated on the 6-well plate (200,000 cells/well). After 24 h, cells were divided into probes per 200,000 cells. Next, cells were prepared and stained following the annexin V kit and protocol (Life Technologies, Invitrogen, USA).
All stained cells were analyzed by Cytoflex Beckman Coulter cytometer (Beckman Coulter Life Sciences, ID, USA). Analysis of the obtained results was performed using FlowJo v10 (FlowJo LLC, USA).
All stained cells were analyzed by Cytoflex Beckman Coulter cytometer (Beckman Coulter Life Sciences, ID, USA). Analysis of the obtained results was performed using FlowJo v10 (FlowJo LLC, USA).
10
Apoptosis and Cell Cycle Analysis
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Apoptotic cell death was assessed using annexin V/PI staining (Yeasen, Shanghai, China). Transfected cells were fixed in 75% ethanol at 4 °C for 12 h and stained with propidium iodide (PI) (Yeasen) solution for cell cycle analysis. The proportion of cells in each cell cycle phase was analyzed using a CytoFlex cytometer (Beckman Coulter, USA). All data were analyzed using the FlowJo V10 software.
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