For cell cycle analysis, transfected A549 cells were harvested, washed three times with cold PBS and fixed with cool 70% ethanol at room temperature. Then the cells were stained with 50 μg/ml propidium iodide (PI, BD Biosciences, CA, USA) following the manufacturer’s protocol for 30 min, followed by cell cycle analysis using a FACS Calibur Flow Cytometer (Beckman Coulter, Atlanta, GA, USA). To measure cell apoptosis, Annexin V-FITC apoptosis detection kit (BD Biosciences, CA, USA) was applied. Briefly, cells were collected, washed twice with cold PBS, and re-suspended in Annexin V-binding buffer. Following incubation with Annexin V-FITC and PI for 15 min in the dark, the early apoptosis rate (Annexin V+/PI-) and late apoptosis rate (Annexin V+/PI+) were analyzed using a FACS Calibur Flow Cytometer (Beckman Coulter, Atlanta, GA, USA).
Facscalibur flow cytometer
Manufactured by Beckman Coulter
Sourced in United States, Taiwan, Province of China
The FACSCalibur flow cytometer is a instrument designed to analyze the physical and chemical characteristics of particles, cells, or other biological samples in a fluid stream. It utilizes laser technology to detect and measure multiple parameters of individual cells or particles passing through the instrument's flow cell.
Automatically generated - may contain errors
Lab products found in correlation
Fitc annexin 5 apoptosis detection kit, by BD (5 mentions)
Propidium iodide, by Merck Group (4 mentions)
Cell counting kit 8 cck 8, by Dojindo Laboratories (3 mentions)
Propidium iodide, by BD (2 mentions)
Dcfh da, by Merck Group (2 mentions)
Cd44 pe, by BD (2 mentions)
Cd133 apc, by BD (2 mentions)
Annexin 5 fitc apoptosis detection kit, by Thermo Fisher Scientific (2 mentions)
Cell quest software, by Beckman Coulter (2 mentions)
Cellquest software, by BD (2 mentions)
Cytofix cytoperm kit, by BD (2 mentions)
Rnase a, by Merck Group (2 mentions)
Kaluza software, by Beckman Coulter (2 mentions)
Pharmingen fitc annexin 5 apoptosis detection kit 1, by BD (2 mentions)
Cycletest plus dna reagent kit, by BD (2 mentions)
Synergy ht, by Agilent Technologies (2 mentions)
Cell quest pro software, by Beckman Coulter (2 mentions)
Annexin 5 fluorescein isothiocyanate fitc propidium iodide staining kit, by Dojindo Laboratories (2 mentions)
Cell cycle kit, by BestBio (2 mentions)
Trypsin, by Merck Group (1 mentions)
98 protocols using facscalibur flow cytometer
1
Cell Cycle and Apoptosis Analysis
2
Measuring Intracellular ROS Levels
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Change in intracellular ROS levels were by measuring the oxidative conversion of cell permeable 2′,7′-dichlorofluorescein diacetate (DCFH-DA, Sigma) to fluorospectro-photometer (Chen et al., 2018 (link)). Cells were plated in 6 cm dish (1 × 106 cells/well) and allowed to attach overnight. Cells were incubated with control media or RSL3 for 24 h. The cells were washed with D-Hank’s and incubated with DCFH-DA at 37°C for 20 min. Then DCF fluorescence distribution of 20,000 cells was detected by FACS Calibur Flow Cytometer (Beckman Coulter, CytoFLEX S, United States) at an excitation wavelength of 488 nm and at an emission wavelength of 535 nm.
3
Isolation of Testicular Cell Populations
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The testes from 8-week-old male mice were removed and decapsulated. Firstly, the seminiferous tubules were incubated in 10 ml 1× PBS containing 90 mg/ml of collagenase (Invitrogen) with continuous agitation for 15 min at 32 °C and then allowed to sediment for 5 min and the supernatant was removed. Next, the pellet was resuspended in 10 ml of PBS with 60 mg/ml of trypsin (Sigma-Aldrich) and 1 µg/ml of DNase (Promega), and incubated under the same conditions for 15 min. After gently being pipetted with a Pasteur pipette, the cell suspension was centrifuged at 400 × g for 10 min and then washed three times with 1× PBS, filtered using 40 µm nylon mesh to remove cell clumps and last resuspended in HEPES-buffered RPMI containing 0.5% BSA. After the testicular single cell suspensions were obtained, two million cells were diluted in 2 ml of 1× PBS buffer and stained with Hoechst 33342 (5 μg/ml; Sigma) for 1 h at 32 °C. Before analysis, PI (2 μg/ml; Sigma) was added to exclude dead cells. Finally, cell analysis and sorting were performed on a FACScalibur flow cytometer (Beckman Coulter, CA, USA) equipped with a cell sorting system.
4
Flow Cytometric Analysis of MSC Markers
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For flow cytometry analysis, 1×10
5cells were used. Cells were incubated for 20 min in a dark condition and room temperature with an appropriate amount of fluorescence conjugated monoclonal antibodies for MSC surface positive (CD105 and CD90) and negative (CD34 and CD45) markers (all from Ebioscience, USA). Subsequently, the cells were washed and centrifuged for 5 min at 800 g, then read on a FACS Calibur flow cytometer (Beckman, Fullerton, CA, USA).
5cells were used. Cells were incubated for 20 min in a dark condition and room temperature with an appropriate amount of fluorescence conjugated monoclonal antibodies for MSC surface positive (CD105 and CD90) and negative (CD34 and CD45) markers (all from Ebioscience, USA). Subsequently, the cells were washed and centrifuged for 5 min at 800 g, then read on a FACS Calibur flow cytometer (Beckman, Fullerton, CA, USA).
5
Quantifying Microglia Phenotypes in Spinal Cord
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The percentage of M1/M2 microglia phenotype was determined by evaluating the levels of their corresponding markers CD16 and CD206. Firstly, rat L4-L6 spinal cord samples were cut with scissors, detached with 0.15% trypsin, and repeatedly blown into single-cell suspension, followed by isolation of mononuclear cells by Percoll density gradient centrifugation. The mononuclear cells were then resuspended in PBS and stained with fluorescence-labeled CD16 (ab246222, Abcam) and CD206 antibodies (ab270647, Abcam) for 30 min at 4 °C in the dark. For cell culture samples, HAPI cells were collected, rinsed with PBS, adjusted to 1 × 106 cells/mL, and stained with the same CD16 (Abcam) and CD206 (Abcam) antibodies. Later, the percentages of CD16 and CD206 were measured by a FACSCalibur flow cytometer equipped with FlowJo software (version vX 0.7) (Beckman Coulter, Brea, CA, USA).
6
Quantifying Cell Apoptosis Rates
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In order to quantify the cell apoptosis rates, a flow Annexin V-FITC Apoptosis Detection kit (BD Biosciences) was applied according to the manufacturer's protocols. In brief, the transfected ESCC cells were collected and resuspended in 500 µl Annexin V-binding buffer (BD Biosciences). Subsequently, 5 µl of FITC-conjugated Annexin V and 5 µl propidium iodide were added. Stained cell fluorescence was analyzed using the FACSCalibur flow cytometer (Beckman Coulter, Inc.) following incubation for 10 min at room temperature in the dark. The results of flow Annexin V-FITC Apoptosis Detection kit were analyzed using FlowJo software (version 9.6.2; FlowJo LLC). The caspase-3/-7 activity apoptosis assay was performed using a caspase-3/-7 Activity Apoptosis Assay kit (https://www.aatbio.com ). ESCC cells were incubated with caspase-3/-7 assay solution (AAT Bioquest) for 1 h at room temperature. Fluorescence intensity was detected at Ex/Em=490/525 nm using a fluorescence microplate reader (Tecan Group, Ltd.).
7
Macrophage Polarization in Glioma Microenvironment
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THP-1 cells were cultured in RPMI-1640 medium with 10% fetal bovine serum and 100 ng/ml Phorbol-12-myristate-13-acetate (PMA) for 72 h. The adherent THP-1 Cells induced by PMA were co-incubated with U87 cells stained with GFP fluorescence for 48 h. The THP-1 cells were then sorted and harvested by a SONY SH800 Cell Sorter. After washed by PBS twice, the sorted cells were incubated with Alexa Fluor® 647-conjugated anti-human CD206, Phycoerythrin-conjugated anti-human CD80, (all 1:100, Abcam). Multiple-color FACS analysis was performed using a FACS Calibur Flow Cytometer (Beckman Coulter, Atlanta, GA, USA) and analyzed by FlowJo software (TreeStar, San Carlos, CA).
8
Quantifying Intracellular Lipid ROS and Mitochondrial Membrane Potential
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Changes in intracellular lipid ROS levels were measured by the oxidative conversion of cell permeable 2′,7′-dichlorofluorescein diacetate (DCFH-DA, Sigma) with a fluorescence spectrophotometer. Cells were plated in 6 cm dishes (1 × 106 cells/well) and allowed to attach overnight. The cells were incubated with control medium with or without sorafenib for 24 h. The cells were washed with D-Hank's solution and incubated with DCFH-DA at 37 °C for 20 min. Then, the DCF fluorescence distribution of 20,000 cells was determined by a FACSCalibur flow cytometer (Beckman Coulter, CytoFLEX S, United States) at an excitation wavelength of 488 nm and at an emission wavelength of 535 nm [14] (link).
The mitochondrial membrane potential (MMP) assay is a cytofluorimetric method that is both qualitative and quantitative, and the results are validated by analysing the MMP at the level of a single mitochondrion. JC-1 in an aggregated form emits a red or orange emission (590 ± 17.5 nm) in the matrix of the mitochondria, indicating a normal MMP; with the loss of MMP, JC-1 is converted to the monomeric form and emits green fluorescence with an emission of 530 ± 15 nm. Therefore, a decrease in the red/green fluorescence intensity ratio indicates a reduction in MMP [15] .
The mitochondrial membrane potential (MMP) assay is a cytofluorimetric method that is both qualitative and quantitative, and the results are validated by analysing the MMP at the level of a single mitochondrion. JC-1 in an aggregated form emits a red or orange emission (590 ± 17.5 nm) in the matrix of the mitochondria, indicating a normal MMP; with the loss of MMP, JC-1 is converted to the monomeric form and emits green fluorescence with an emission of 530 ± 15 nm. Therefore, a decrease in the red/green fluorescence intensity ratio indicates a reduction in MMP [15] .
9
Apoptosis Analysis via Flow Cytometry
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Cell apoptosis analysis was conducted with propidium iodide and Annexin V-fluorescein isothiocyanate (Beijing Biosea Biotechnology, Beijing, China). Briefly, 2×105 cells were seeded into 6-well plates for 24 h. Cells were collected and washed with phosphate buffer saline, then stained cells with propidium iodide-staining solution and Annexin V-fluorescein isothiocyanate. After 30 min, cell fluorescence was examined with a FACS Calibur flow cytometer (Beckman Coulter, Fullerton, CA, USA).
10
Annexin V-FITC Membrane Integrity Assay
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Annexin V-FITC staining kit was used to determine membrane integrity and phosphatidylserine (PS) externalization [66 (link)]. Cells (106) were grown in 35 mm diameter plates, annexin V-FITC (10 μg/mL) and PI (20 μg/mL) were used to label the cells. A binding buffer was used to wash all plates and then the cells were harvested. A binding buffer was used to suspend the cells (2 × 105 cells/mL) and the label was assessed using a FACS-Caliburflow cytometer (Beckman Coulter, Taipei, Taiwan). The results were analyzed with CellQuest software (BD CellQuest Pro, Franklin Lakes, NJ, USA). Approximately 10,000 cells were counted for each measurement.
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