GraphPad Prism software (v9) was used when performing statistical analysis and in the creation of graphs. Where the unpaired Student’s t-test was performed for statistical analysis: **** for P<0.0001, *** for P<0.001, ** for P<0.01 and * for P<0.05 were considered statistically significant and were indicated in the related figure legends and graphs. Where n.s. is indicated, the P value is not significant. The results are shown as mean ± standard deviation (SD). CytoFlex software (Beckman Coulter) was used when performing analysis of flow cytometric data. QuPath: Open source software for digital pathology image analysis was used to perform colony quantification [23 (link)]. Empiria Studio® Software (LI-COR) was used to quantify western blot bands and normalise bands to total protein.
Cytoflex software
Manufactured by Beckman Coulter
Sourced in United States
CytoFLEX software is a data acquisition and analysis application used with the CytoFLEX flow cytometer. It provides functionality for controlling the instrument, displaying real-time data, and performing basic data analysis.
Automatically generated - may contain errors
Lab products found in correlation
Cytoflex, by Beckman Coulter (3 mentions)
Empiria studio software, by LI COR (1 mentions)
Mitosox, by Thermo Fisher Scientific (1 mentions)
Apc conjugated anti human cd3 antibody, by BD (1 mentions)
M20036, by Thermo Fisher Scientific (1 mentions)
Uchl1, by BioLegend (1 mentions)
Dreg 56, by BioLegend (1 mentions)
Cytoflex cell analyzer, by Beckman Coulter (1 mentions)
Annexin 5 fitc, by BD (1 mentions)
F4 80 apc, by Thermo Fisher Scientific (1 mentions)
Cd25 apc, by Thermo Fisher Scientific (1 mentions)
Cd11b fitc, by Thermo Fisher Scientific (1 mentions)
Foxp3 pe, by Thermo Fisher Scientific (1 mentions)
Cytoflex s flow cytometer, by Beckman Coulter (1 mentions)
Permeabilization buffer, by Thermo Fisher Scientific (1 mentions)
Cd4 percp cyanine5, by Thermo Fisher Scientific (1 mentions)
Mitotracker red, by Thermo Fisher Scientific (1 mentions)
Malvern nano zs90, by Malvern Panalytical (1 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Annexin 5 apc 7 aad apoptosis kit, by MultiSciences Biotech (1 mentions)
17 protocols using cytoflex software
1
Statistical Analysis and Visualization
2
Quantifying Cellular Oxidative Stress
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ROS levels were analyzed according to the reagent manufacturer’s instructions. Isolated splenocytes were treated with 10 µM DCF-DA (Invitrogen, Carlsbad, CA, USA) or 5 µM MitoSOX (Invitrogen, Carlsbad, CA, USA) at 37 °C for 30 min in the dark and then washed twice with DPBS. The ROS population was gated, and a histogram was created. The mean fluorescence intensity (MFI) was obtained using CytoFLEX software (Beckman Coulter, California, USA).
3
Apoptosis Assay of Raji Cells
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Raji cells (1.6 × 105 /well) were divided into three treatment groups, given CAR-T cells (2 × 104 /well) with or without GA (100 μM), GA (100 μM) and the control (PBS) group (n = 3). After incubation for 12 hours, the cells were stained with APC-conjugated anti-human CD3 antibody (BD Pharmingen, USA) and FITC-conjugated Annexin V (Gene-Protein Link, China) for half an hour. The apoptosis rate of Raji cells was then measured by flow cytometric analysis (CytoFLEX, Beckman Coulter, USA), and FACS data was analyzed by CytoFLEX Software (Beckman Coulter, USA) and FlowJo (Version 10.0.7).
4
Analyzing Mitochondrial Membrane Potential
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For each sample, the cells were re‐suspend in cell culture medium or PBS at approximately 1 × 106 cells/ml. For the control samples, 1 μl of 50 mM CCCP was added to the cells and incubated for 5 min at 37°C, 5% CO2. Experimental samples had 1 μl of 20 μM stock TMRM (M20036, Thermo Scientific) reagent (20 nM final concentration) added and were incubated for 30 min at 37°C. Cells were washed once in 1 ml of PBS and then re‐suspended in 500 μl of PBS. The cells were analyzed on a CytoFLEX software (Beckman Coulter, USA) with 561‐nm excitation, using emission filters appropriate for R‐phycoerythrin.
5
Analyzing CAR-T Cell Proliferation
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CAR-T cells were pre-labeled with the intracellular fluorescent label carboxyfluorescein diacetate succinimidyl ester. Covalently bound carboxyfluorescein diacetate succinimidyl ester is divided equally between daughter cells, enabling the discrimination of successive cell division rounds. After culturing labeled CAR-T cells with Raji cells at an E/T ratio of 1:2 for 24 hours, samples were analyzed by flow cytometry analysis by staining with APC labeled anti-human CD3 antibody (clone HIT3a, BioLegend). The proliferation assays and carboxyfluorescein diacetate succinimidyl ester-staining were performed in the continuous presence of IL-2.
FITC-labeled anti-human CD4 antibody (clone OKT4, BioLegend) and PerCP-Cyanine5.5-labeled anti-human CD8 antibody (clone SK1, BioLegend) were used to detect CD4+/CD8+ T cells by FLOW CYTOMETRY ANALYSIS.
PE/Cyanine7-labeled anti-human CD45RO antibody (UCHL1, BioLegend) and PE-labeled CD62L antibody (DREG-56, BioLegend) were used to differentiate memory and effector T cell populations; central memory T (TCM) cells were defined as cells expressing CD45RO and CD62L.
Flow cytometry analysis was conducted on a CytoFLEX Cell Analyzer (Beckman Coulter), and data were analyzed using CytoFLEX Software (Beckman Coulter).
FITC-labeled anti-human CD4 antibody (clone OKT4, BioLegend) and PerCP-Cyanine5.5-labeled anti-human CD8 antibody (clone SK1, BioLegend) were used to detect CD4+/CD8+ T cells by FLOW CYTOMETRY ANALYSIS.
PE/Cyanine7-labeled anti-human CD45RO antibody (UCHL1, BioLegend) and PE-labeled CD62L antibody (DREG-56, BioLegend) were used to differentiate memory and effector T cell populations; central memory T (TCM) cells were defined as cells expressing CD45RO and CD62L.
Flow cytometry analysis was conducted on a CytoFLEX Cell Analyzer (Beckman Coulter), and data were analyzed using CytoFLEX Software (Beckman Coulter).
6
Annexin V-FITC Apoptosis Assay
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To detect the induction of apoptosis, treated HK2 cells were washed twice with cold PBS, incubated with 10 µg/ml Annexin V-FITC (BD) for 15 min at room temperature, and subsequently propidium iodide (PI; 10 µl) was added and incubated for 5 min at room temperature. The samples were analyzed using CytoFlex software (version 2.0; Beckman Coulter, Inc.).
7
Murine Liver Lymphocyte Isolation and Analysis
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Mice liver was harvested and rendered into single cell suspensions, and lymphocytes were isolated and purified by density-gradient centrifugation on Percoll-PBS separation medium (GE Healthcare, Sweden). Then the isolated lymphocytes were washed twice in PBS and conserved in 0.1% BSA. Cells were incubated with CD45-v450 (1 × 107 cells suspension in 100 μL 1% BSA with 10 μL antibody, eBioscience, USA), F4/80-APC (eBioscience), CD11b-FITC (eBioscience) to analyze macrophage. 1 × 106 cells were incubated with CD3-APC-Cyanine7 (eBioscience), CD4-Percp-Cyanine5.5 (eBioscience), CD25-APC (eBioscience) to analyzed Treg. After 30 min antibody incubation at room temperature, protect from light. After two times washing, the cells were fixed with 1 mL of Foxp3 Fixation (eBioscience) working solution for 60 min at room temperature, protect from light. Then cells were washed twice by Permeabilization Buffer (eBioscience), the lymphocytes were incubated with Foxp3-PE (eBioscience) for 40 min at room temperature, protect from light. The lymphocytes were then detected with a CytoFLEX S Flow cytometer (Beckman, USA) and analyzed using CytoFLEX software (Beckman).
8
Characterization of Isolated Mitochondria
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The sizes and zeta potentials of the isolated mitochondria were measured using a Malvern Nano-ZS 90 laser analyzer (Malvern Instruments, Malvern, UK), and the mitochondrial concentrations were analyzed with a Pierce BCA protein assay kit (Thermo Fisher, Waltham, MA, USA). Purity of isolated mitochondria from stained L6 cells by Mitotracker Red (Molecular Probes, Eugene, OR, USA) were analyzed using CytoFLEX software (Beckman Coulter, California, USA). Other organelles in isolated mitochondria were detected using Western blot analysis.
9
Apoptosis Analysis via Annexin V-APC/7-AAD Assay
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Cell apoptosis was analyzed by the Annexin V-APC/7-AAD apoptosis kit (MULTI Science, Hangzhou, China) according to the instructions of the kit. After transfection, shRNA-HT29 cells and shNEAT1-HT29 cells were stained with Annexin V-APC and 7-AAD. The flow cytometry was performed using a Beckman CytoFLEX, and the data were analyzed using CytoFLEX Software (Beckman Coulter, California, America). Image J software was used to count the apoptosis cells, and the data was performed with independent sample t test using SPSS 25.0.
10
Apoptosis and Cell Death Assay
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At predetermined time
points (24, 72, and 120 h post-treatment), H9C2 and SAOS-2 (at pH
7.4 and 6.0) were trypsinized and incubated with annexin V-fluorescein
isothiocyanate (FITC)/propidium iodide (PI) (Thermo Fisher) for viability,
apoptosis, and cell death assessment by flow cytometry. Briefly, at
each time point, cells were washed with Dulbecco’s phosphate-buffered
saline solution without calcium and magnesium and detached using trypsin–ethylenediaminetetraacetate
(0.05%). A total of 100 × 103 cells per tube were
stained with 2.5 μM annexin V-FITC and 1 μg/mL PI in annexin
V binding buffer (1×) for 15 min at 37 °C protected from
light. Cells stained with annexin V-FITC/PI were evaluated using a
Beckman Coulter cytoflex. Live cells (annexin V–/PI−),
apoptotic cells (annexin V+/PI−), and dead cells (PI+) were
analyzed using Cytoflex software (Beckman Coulter). Untreated cells
were used as controls.
points (24, 72, and 120 h post-treatment), H9C2 and SAOS-2 (at pH
7.4 and 6.0) were trypsinized and incubated with annexin V-fluorescein
isothiocyanate (FITC)/propidium iodide (PI) (Thermo Fisher) for viability,
apoptosis, and cell death assessment by flow cytometry. Briefly, at
each time point, cells were washed with Dulbecco’s phosphate-buffered
saline solution without calcium and magnesium and detached using trypsin–ethylenediaminetetraacetate
(0.05%). A total of 100 × 103 cells per tube were
stained with 2.5 μM annexin V-FITC and 1 μg/mL PI in annexin
V binding buffer (1×) for 15 min at 37 °C protected from
light. Cells stained with annexin V-FITC/PI were evaluated using a
Beckman Coulter cytoflex. Live cells (annexin V–/PI−),
apoptotic cells (annexin V+/PI−), and dead cells (PI+) were
analyzed using Cytoflex software (Beckman Coulter). Untreated cells
were used as controls.
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