JC‐1 assay was performed to assess Mitochondrial membrane potential (MMP). 400 µl of JC‐1 (1×) working dilution was added to 100 µl cell suspension and incubated for 15 min at a cell culture incubator. Samples were washed once in PBS, resuspended in complete medium, and analyzed on a Coulter XL Flow Cytometer equipped with an Argon laser (Beckman Coulter) JC‐1 fluorescence was analyzed examining dual parameter FL1(JC‐1 Green) versus FL2 (JC‐1 Red) histograms controlling for intact cells (based on size criteria). Cells exhibiting loss of mitochondrial potential display progressively increased JC‐1 green fluorescence with eventual loss of JC‐1 red fluorescence. Increases in JC‐1 Red/JC‐1 Green co‐positive and JC‐1 Green positive alone events relative to control samples represent increased loss of mitochondrial potential.
Coulter xl flow cytometer
Manufactured by Beckman Coulter
Sourced in United States
The Coulter XL Flow Cytometer is a laboratory instrument designed for the analysis of cells and particles. Its core function is to rapidly detect, identify, and quantify various characteristics of cells or particles suspended in a fluid, such as size, granularity, and fluorescence. The Coulter XL utilizes principles of flow cytometry to provide high-speed, multiparametric analysis of individual cells or particles.
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Lab products found in correlation
Expo32 adc, by Beckman Coulter (1 mentions)
Pi rnase staining solution, by BD (1 mentions)
Modfit 3, by Verity Software House (1 mentions)
Fitc annexin 5 apoptosis detection kit 1, by BD (1 mentions)
Fc block 2.4g2, by BD (1 mentions)
Cell fit software, by BD (1 mentions)
Propidium iodide, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Trypsin, by Thermo Fisher Scientific (1 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
Afatinib, by Boehringer Ingelheim (1 mentions)
Microplate reader 550, by Bio-Rad (1 mentions)
Cetuximab, by Merck Group (1 mentions)
5 protocols using coulter xl flow cytometer
1
Assessing Mitochondrial Membrane Potential
2
Apoptosis and Cell Cycle Analysis After Radiation Exposure
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Cells were stained with the Annexin V/FITC Apoptosis Detection Kit I (BD Pharmingen, Franklin Lakes, NJ, USA) 48 h after receiving a 10 Gy radiation dose to look for signs of apoptosis. Using a Coulter-XL flow cytometer (Beckman Coulter Inc., Brea, CA, USA) and an EXPO32 ADC, we were able to capture and analyze pictures of apoptosis. The total apoptosis rate of tumor cells was calculated by adding the apoptosis rates observed in the early (lower-right) and late (upper-right). After 24 h, cells that had been irradiated with 10 Gy were collected and preserved in cold ethanol (1 mL PBS + 2 mL absolute ethanol) for studying the cell cycle. The cells were stained with a PI/RNase staining solution (BD Pharmingen; Franklin Lakes, NJ, USA). After that, we analyzed the cells cycles distributions (G1 and G2 phase) using flow cytometry (Coulter-XL; Beckman Coulter Inc., Brea, CA, USA) and Mod Fit 3.0 (Verity Software House, Inc., USA).
3
Isolation and Characterization of Immune Cells from Mouse Tissues
4
Cell Cycle Analysis of A549 Cells Treated with RTHF
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A549 cells were cultured in Roswell Park Memorial Institute (RPMI) (Hyclone, Logan, UT, USA) supplemented with 10% fetal bovine serum (Logan) in a humidified incubator containing 5% CO2 in air at 37°C, and subculture was carried out with 0.25% trypsin–0.02% ethylene diamine tetraacetic acid (EDTA) (Logan). The RTHF was dissolved in phosphate-buffered saline (PBS) and adjusted to a final concentration of 10 mg/mL.
First, A549 cells (1×105 cells/mL) were treated with various concentrations of RTHF (0–64 μg/mL) for different time points (24, 48, and 72 h). The A549 cells were then treated with 3 μg/mL of RTHF for 48 h. All the cell samples were then harvested, fixed in 70% ethanol (Merck KGaA, Darmstadt, Germany) and stored at −20°C for 24 h until further analysis. Next, the cells were washed twice with ice-cold PBS and then incubated with RNase and propidium iodide (PI) (Sigma-Aldrich Co., St Louis, MO, USA) for 30 min.
The PI-stained cells were excited at a wavelength of 488 nm and emitted a maximum wavelength of 617 nm. Acquisition of 10,000 events was chosen to measure the distribution of cells in the different cell cycle phases using a Coulter XL Flow Cytometer (Beckman Coulter Inc, Brea, CA, USA). The distribution of cells in the different cell cycle phases shown in the DNA histograms was analyzed using the Becton Dickinson Cell Fit Software (BD, Franklin Lakes, NJ, USA).
First, A549 cells (1×105 cells/mL) were treated with various concentrations of RTHF (0–64 μg/mL) for different time points (24, 48, and 72 h). The A549 cells were then treated with 3 μg/mL of RTHF for 48 h. All the cell samples were then harvested, fixed in 70% ethanol (Merck KGaA, Darmstadt, Germany) and stored at −20°C for 24 h until further analysis. Next, the cells were washed twice with ice-cold PBS and then incubated with RNase and propidium iodide (PI) (Sigma-Aldrich Co., St Louis, MO, USA) for 30 min.
The PI-stained cells were excited at a wavelength of 488 nm and emitted a maximum wavelength of 617 nm. Acquisition of 10,000 events was chosen to measure the distribution of cells in the different cell cycle phases using a Coulter XL Flow Cytometer (Beckman Coulter Inc, Brea, CA, USA). The distribution of cells in the different cell cycle phases shown in the DNA histograms was analyzed using the Becton Dickinson Cell Fit Software (BD, Franklin Lakes, NJ, USA).
5
Lung Adenocarcinoma Cell Line A549
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The lung adenocarcinoma cell line A549 was obtained from the Shanghai Cell Institute of the Chinese Academy of Sciences. RPMI 1640 medium, fetal bovine serum, and trypsin were purchased from Gibco Company (Grand Island, NY, USA). SPB was purchased from Merck, (Kenilworth, NJ, USA). Afatinib was from Boehringer Ingelheim (Ingelheim, Germany). Cetuximab was from Merck. Penicillin was obtained from Beijing Chemical Factory (Beijing, China). Streptomycin was from PeproTech (Rocky Hill, NJ, USA). The 5% tetrazolium blue solution (MTT solution) was from Shanghai Sino-American Biotechnology Company (Shanghai, China). The Coulter XL flow cytometer was obtained from Beckman Coulter (Brea, CA, USA). The 550 microplate reader was purchased from Bio-Rad Inc. (Hercules, CA, USA). The CKX4 biological inverted microscope was obtained from Olympus (Tokyo, Japan).
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