Cells were treated with MRZ for 24 hours. Prior to collection, the cells were stained for superoxide and general oxidative stress using the Cellular ROS/Superoxide Detection Assay Kit (Abcam). Stained cells were then assayed by flow cytometry on the Gallios 561 (Beckman Coulter), and the Kaluza acquisition software (Beckman Coulter).
Kaluza acquisition software
Manufactured by Beckman Coulter
Sourced in United States
Kaluza Acquisition Software is a data acquisition and analysis software solution developed by Beckman Coulter. It is designed to work with Beckman Coulter's flow cytometry instruments, enabling users to collect and analyze cell population data.
Automatically generated - may contain errors
Lab products found in correlation
Gallios flow cytometer, by Beckman Coulter (6 mentions)
Gallios 561, by Beckman Coulter (1 mentions)
Cellular ros superoxide detection assay kit, by Abcam (1 mentions)
Dna prep coulter reagent kit, by Beckman Coulter (1 mentions)
Anti sca 1, by Miltenyi Biotec (1 mentions)
Anti cd31 antibody, by Miltenyi Biotec (1 mentions)
Anti cd117, by Miltenyi Biotec (1 mentions)
Lipopolysaccharides (lps), by Hycult Biotech (1 mentions)
Immunoprep reagent system, by Beckman Coulter (1 mentions)
7 protocols using kaluza acquisition software
1
Oxidative Stress Quantification in Cells
2
Cell Cycle Analysis by Flow Cytometry
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Cells were detached with trypsin, harvested and washed with 1X PBS (phosphate‐buffered saline), fixed in 70% ethanol, and stored at −20 °C. Before the analysis, fixed cells were centrifuged (1252 g , 5 min) and incubated for 3 min at 37 °C in PBS. After centrifugation, cell pellets were dissociated and incubated with RNAse and propidium iodide using the DNA‐Prep Coulter Reagent kit (Beckman Coulter, Villepinte, France) and were analyzed with a Gallios flow cytometer (Beckman Coulter). A computerized gating was applied on the side and front diffusion to exclude small debris and on a pulse width and an integral red fluorescence peak to remove aggregates. The data were analyzed by the Kaluza® acquisition software (Beckman Coulter).
3
Erythrocyte Phosphatidylserine Exposure Assay
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At the time of sacrifice, 30 uL of the whole blood collected via cardiac puncture was fixed in 100 uL of a solution containing 1% formalin and 0.01% glutaraldehyde in PBS, and stored at 4 °C until use. To assess PS exposure, 10 uL of the fixed cell suspension was aliquoted into new centrifuge tubes, washed with PBS, and blocked in 3% BSA for 1 h. Fixed cells were then resuspended in a 3% BSA PBS solution containing a 1:500 dilution of both anti-Ter119 conjugated to APC/Cy7 antibody (Biolegend; Cat# 116223) and Annexin-PE (BD Pharmingen; Cat# 556421), and incubated for 30 min at room temperature protected from light. Cells were washed with PBS and analyzed using a Gallios flow cytometer (Beckman Coulter, Indianapolis, IN, Model #B5-R1-V2). 50,000 events were recorded for each sample. Dual labeling of Ter119 and Annexin was detected and expressed as percentage of total population of cells. Quadrant gates were set based on unlabeled and singly labeled samples, and the gates were applied universally to each sample. Log scale was used for both Ter119 and Annexin V. No compensation was used. Data was analyzed with Kaluza Acquisition Software (Beckman, Coupter, Indianapolis, IN).
4
Phenotypic Analysis of Cultured hCSCs
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Cultivated hCSCs were harvested by centrifugation after treatment with trypsin and subsequently stained with PE-coupled anti-CD105, anti-CD117, anti-Sca1 or anti-CD31 antibody (Miltenyi Biotec, Bergisch Gladbach, Germany) according to manufacturer’s guidelines. For isotype controls, hCSCs were stained with PE-coupled IgG1 control antibody or APC-coupled IgG1 control antibody. Analysis was done using Gallios Flow Cytometer (Beckmann Coulter Inc., Brea, CA, USA), while Kaluza Acquisition Software (Beckmann Coulter Inc.) was used for subsequent data acquisition and statistical analysis.
5
Platelet Activation and Flow Cytometry Analysis
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Platelet activation was determined by the increase in mean fluorescence binding intensity (MFI) of the activation marker P-selectin (CD62P) and PAC-1 (activated GPIIb/IIIa) on CD61 gated washed human platelets on a Gallios Flow Cytometer by means of the Kaluza Acquisition Software (Beckman Coulter Life Sciences, Indianapolis USA) on least 50’000 CD61+ events. Cutoff levels on unstimulated platelets were determined by a 95% confidence interval and results presented in box and whisker plot using GrapPad Prism V 6.07 (GraphPad Software, Inc. La Jolla, CA 92037 USA).
6
CD38 Expression in T-Cell Subsets
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The expression of CD38 was evaluated in CD4 + and CD8 + T-cell subsets by flow cytometry in 100μL fresh anticoagulated whole blood. The cells were labeled with the following antibodies: anti-CD38-APC-Cyanine 5.5 (APC-Cy5.5, clone HIT2, Invitrogen, Frederick, MD), anti-CD4-APC-Cyanine 7 (APC-Cy7, clone OKT4, BioLegend, San Diego, CA), anti-CD8-Pacific Blue (PB, clone SK1, BioLegend, San Diego, CA), anti-CD3-Pacific Orange (PO, clone VCHT1, Invitrogen, Frederick, MD) and incubated for 20 min at room temperature in the dark. Next, the IMMUNOPREP Reagent System (Beckman Coulter, Mervue Galway, Ireland) was added to each sample using a Coulter MULTI-Q-PREP Lysing Workstation (Beckman Coulter, Miami, FL) to lyse and fixate them.
Fluorescence was measured with a Gallios™ flow cytometer (Beckman Coulter, Miami, FL). The number of events was stopped at a minimum of 200,000 cells in the lymphocyte gate for each sample and flow cytometry data were analyzed using Kaluza™ acquisition software (version 1.5; Beckman Coulter, Miami, FL). (Raybiotech, Georgia, USA), LPS (HycultBiotech, Uden, The Netherlands), LBP (R&D Systems, Minneapolis, USA) and FABP2 (Raybiotech, Georgia, USA), which were performed according to the manufacturer's procedure for each specific commercial ELISA.
Fluorescence was measured with a Gallios™ flow cytometer (Beckman Coulter, Miami, FL). The number of events was stopped at a minimum of 200,000 cells in the lymphocyte gate for each sample and flow cytometry data were analyzed using Kaluza™ acquisition software (version 1.5; Beckman Coulter, Miami, FL). (Raybiotech, Georgia, USA), LPS (HycultBiotech, Uden, The Netherlands), LBP (R&D Systems, Minneapolis, USA) and FABP2 (Raybiotech, Georgia, USA), which were performed according to the manufacturer's procedure for each specific commercial ELISA.
7
Cell Cycle Analysis by Flow Cytometry
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A549 cells were seeded into 6-well cell culture plates at a density of 1 x 10 5 cells per well in complete cell culture medium and underwent cell synchronization as described above. Thereafter, cells were harvested from the plate using trypsin/EDTA, resuspended in phosphate-buffered saline (PBS) and fixed with 70% precooled ethanol for 2 h at 4°C. After a washing step in PBS, cells were stained with propidium iodide (PI) staining solution (10 µg/ml PI, 0.1% Triton X-100, 100 µg/ml DNase-free RNase A in PBS) for 30 min at room temperature (RT). Cell cycle was analyzed by flow cytometry using the Gallios™ flow cytometer and Kaluza acquisition software (Beckman Coulter, Krefeld, Germany). Upon excitation with a 488 nm laser, PI signals were collected in the FL3 channel (630 nm with 30 nm band pass width). Data were analyzed with Kaluza analysis software.
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