Flow cytometry analysis was performed to determine the fraction of G0/G1, S and G2/M phases of FTC-133 cells. Briefly, the cells were transfected with miR-199a-5p mimics or its inhibitor, or CTGF siRNA, and then harvested by trypsinization. Cold 75% ethanol was used to fix cells overnight at 4°C.We harvested the fixed cells, washed them twice with PBS, and resuspended them in PBS. Next, 10 ul of RNaseA was added and mixed, and then 25 ul of PI (propidium iodide) was added and incubated for 30 min in a 37°C water bath. Finally, we filtered the cells once prior to being loaded into the flow cytometry machine (Beckman Coulter).
Flow cytometry machine
Manufactured by Beckman Coulter
Sourced in United States
The flow cytometry machine is a laboratory instrument used for the analysis and sorting of cells or other particles suspended in a fluid stream. It measures and analyzes multiple physical characteristics of individual particles, including size, granularity, and fluorescence, as they pass through a laser beam. The core function of the flow cytometry machine is to rapidly analyze and categorize a large number of particles in a heterogeneous sample.
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Lab products found in correlation
Annexin 5 fitc, by BD (2 mentions)
Propidium iodide, by BD (2 mentions)
Ethylene diamine tetraacetic acid (edta), by Thermo Fisher Scientific (2 mentions)
Annexin 5 fitc pi double staining, by Nanjing Jiancheng (1 mentions)
Bodipy 581 591 c11 probe, by Thermo Fisher Scientific (1 mentions)
Annexin 5 pe kit, by BD (1 mentions)
Ros detection kit, by Beyotime (1 mentions)
Cd86 fitc, by BioLegend (1 mentions)
F4 80 apc, by Thermo Fisher Scientific (1 mentions)
Cd86 pe, by BioLegend (1 mentions)
Cd11b bv510, by BioLegend (1 mentions)
Trypsin, by Thermo Fisher Scientific (1 mentions)
7 protocols using flow cytometry machine
1
Cell Cycle Analysis by Flow Cytometry
2
Analyzing Cell Apoptosis via Annexin V-FITC/PI Staining
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Annexin V-FITC/PI double staining (G003-1-2; Nanjing Jiancheng Bioengineering Institute, Jiangsu, China) was used to stain the cells, and flow cytometry was used to detect cell apoptosis. Annexin V is a sensitive indicator of early cell apoptosis. Propidium iodide (PI) is a nucleic acid dye that stains dead cells and cells in the metaphase and advanced stages of apoptosis. The SSCs were collected by centrifugation and suspended in a 1× binding buffer to adjust the cell concentration to 1 × 106/mL. Annexin V-FITC (5 μL) and PI (10 μL) were added to the tubes and mixed gently. After incubation, the samples were placed in an ice bath in the dark and then analyzed by a flow cytometry machine (Beckman, CA, USA).
3
Apoptosis and Oxidative Stress Evaluation
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The PE Annexin V Kit (BD, 559763, USA) was utilized to measure the apoptosis status of the cells. The tested cells were collected into centrifuge tubes and incubated with 7AAD and Annexin V dye for 15 min at 25°C. The prepared cells were identified by flow cytometry machine (Beckman, USA) with PE and PerCP fluorescence channel. Reactive Oxygen Species (ROS) detection kit (Beyotime, S0033S, China) was utilized to measure ROS in the cells. The BODIPY 581/591 C11 probe (Invitrogen, D3861, USA) was used to measure intracellular lipid peroxidation. The cells were incubated for 30 min after adding the probe. A flow cytometry machine with a FITC channel was used for evaluation. All results were analyzed using FlowJo software version 10.8.1.
4
Annexin V-FITC and PI Apoptosis Assay
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Following the applied genetic treatments, Annexin V-FITC and Propidium Iodide (PI) dyes (each at 10 μg/mL, BD Pharmingen, San Diego, CA) were added for 30 min under the dark at room temperature. Cell apoptosis was analyzed by a flow cytometry machine (Beckman Coulter, Brea, CA).
5
Annexin V-FITC and PI Apoptosis Assay
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Following the applied genetic treatments, Annexin V-FITC and Propidium Iodide (PI) dyes (each at 10 μg/mL, BD Pharmingen, San Diego, CA) were added for 30 min under the dark at room temperature. Cell apoptosis was analyzed by a flow cytometry machine (Beckman Coulter, Brea, CA).
6
Phenotypic Characterization of Activated Macrophages
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Macrophages received medium with the addition of 100 ng/mL LPS and 20 ng/mL IFN‐γ for 48 h. After cell maturation, 0.25% trypsin with 0.02% EDTA (Gibco, Grand Island, USA) was added, and 12‐well were plates into the incubators for 10 minutes during digestion. DMEM with 10% FBS was added to terminate digestion, the cells were placed into a centrifuge at 300g × 5 minutes, and the supernatant was discarded. Cells were resuspended in flow cytometry liquid and blocked with 20 μL rat serum. PE‐F4/80 antibody, positive control antibody and FITC‐CD86 (BioLegend, San Diego, CA) were added at 1:200. The samples were mixed thoroughly and incubated at 4°C for 1 hour. Then, they were washed with flow cytometry liquid twice and assessed on a flow cytometry machine (Beckman Coulter, CA, USA).
7
Polarization and Flow Cytometry of BMDMs
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BMDMs were cultured in vitro with M-CSF-conditioned medium. After culturing for 7 days, BMDMs were stimulated for 48 hours with PBS and 100 ng/mL LPS plus 20 ng/mL IFN‐γ to polarize them into M0 and M1 macrophages, respectively. After cell maturation, 0.25% trypsin with 0.02% EDTA (Gibco, Grand Island, USA) was added, and 6‐well plates were plated into incubators for 2 minutes during digestion. Then, DMEM with 10% FBS was added to terminate digestion, the cells were placed into a centrifuge at 300 × g for 5 minutes, and the supernatant was discarded. Cells were resuspended in flow cytometry liquid and blocked with 20 μL of rat serum. Next, 40 μL of the primary antibody mix (BV510-CD11b, 1:200, BioLegend, 101245; APC-F4/80, 1:50, eBioscience, 17-4801-82; PE-CD86, 1:200, BioLegend, 105014) was added and the cells were incubated for 15 minutes on ice in the dark. Then, the cells were washed twice with flow cytometry liquid and assessed on a flow cytometry machine (Beckman Coulter, CA, USA).
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