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Flow cytometry analysis software

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FlowJo is a flow cytometry analysis software that provides a comprehensive platform for the visualization, analysis, and interpretation of flow cytometry data. It offers a suite of tools to help researchers and scientists effectively manage and analyze their flow cytometry experiments.

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Lab products found in correlation

Lsrfortessa, by BD (2 mentions) Foxp3 transcription factor staining buffer set, by Thermo Fisher Scientific (2 mentions) Facscanto, by BD (2 mentions) Facscalibur, by BD (1 mentions) Annexin 5 fitc apoptosis detection kit, by Beyotime (1 mentions) Binding buffer, by Beyotime (1 mentions) Fitc conjugated goat anti mouse igg antibody, by Thermo Fisher Scientific (1 mentions) Facscanto flow cytometer, by BD (1 mentions) Facsaria 3 flow cytometer, by BD (1 mentions) Cytoperm permeabilization buffer plus, by BD (1 mentions) Cytofix cytoperm buffer, by BD (1 mentions) Apc brdu flow kit, by BD (1 mentions) Bim c34c5, by Cell Signaling Technology (1 mentions) Pe annexin 5 apoptosis detection kit, by BD (1 mentions) Statview 5, by SAS Institute (1 mentions) Jc 1 dye buffer, by Beyotime (1 mentions) Fc500 mpl, by Beckman Coulter (1 mentions) Rpmi 1640 cell culture medium, by Thermo Fisher Scientific (1 mentions) Facsverse, by BD (1 mentions)

10 protocols using flow cytometry analysis software

1

Flow Cytometry Cell Preparation

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Cells were prepared for flow cytometry as in Bellay et al. (2011) (link). In brief, harvested cells were fixed in 70% ethanol for at least 15 min at room temperature, washed with water, and incubated in 0.2 mg/ml RNaseA (BioShop) in 50 mM Tris, pH 8.0, for 2 h at 37°C. Samples were then resuspended in 50 mM Tris, pH 7.5, containing 2 mg/ml proteinase K (BioShop), incubated at 50°C for 40 min, and resuspended in FACS buffer (200 mM Tris, pH 7.5, 200 mM NaCl, and 78 mM MgCl2). Cells were stained with 2× SYBR Green (Life Technologies) in 50 mM Tris, pH 7.5. The samples were briefly sonicated and analyzed by using a Becton Dickinson FACSCalibur. Data were analyzed by using FlowJo Flow Cytometry Analysis Software and plotted on a linear scale unless otherwise stated.
Sing T.L., Hung M.P., Ohnuki S., Suzuki G., San Luis B.J., McClain M., Unruh J.R., Yu Z., Ou J., Marshall-Sheppard J., Huh W.K., Costanzo M., Boone C., Ohya Y., Jaspersen S.L, & Brown G.W. (2018). The budding yeast RSC complex maintains ploidy by promoting spindle pole body insertion. The Journal of Cell Biology, 217(7), 2445-2462.
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2

Apoptosis Analysis of HepG2 Cells

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The apoptosis analysis of HepG2 cells induced by the tested compound was accomplished by flow cytometry using Annexin V-FITC Apoptosis Detection Kit (Beyotime, C1062L) according to the manufacturer’s instructions (18 (link), 19 (link)). Briefly, HepG2 cells were harvested and seeded in 12-well plates (1 × 105 cells/well) and allowed to adhere for 24 h at 37°C. Then, the cells were treated with various concentrations (5, 10, and 15 μM) of casearlucin A. After 48 h incubation, the cells were washed twice with PBS and resuspended in the binding buffer (Beyotime, Shanghai, China). This suspension was incubated for 20 min at room temperature in the dark after adding 10 μL Annexin V-FITC and 5 μL PI. Then, cell apoptosis was examined by BD LSRFortessa flow cytometry (BD Biosciences). The cell apoptosis data were obtained with FLOWJO flow cytometry analysis software (FLOWJO LLC, Ashland, OR, USA).
Li Y., Ma J., Song Z., Zhao Y., Zhang H., Li Y., Xu J, & Guo Y. (2021). The Antitumor Activity and Mechanism of a Natural Diterpenoid From Casearia graveolens. Frontiers in Oncology, 11, 688195.
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3

Foxp3+ Treg Cell Identification

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Cell pellets containing lymphocytes from tissue preparations were resuspended in PBS solution (containing 0.2% BSA and 0.1% NaN3) at a concentration of 1 × 105 – 1 × 106 white blood cells/ml, and 100 µl of the cells were diluted 1:1 with 100 µl of the appropriate monoclonal antibody and incubated at 4°C for 30 min. Foxp3+ Treg cells were identified by staining lymphocytes with anti-CD3, anti-CD4, and anti-CD25, followed by permeabilization using fixation/permeabilization buffer, per the manufacturer’s instructions. Cells were then stained using an anti-Foxp3 antibody. After staining, cells were washed twice with PBS solution (0.2% BSA and 0.1% NaN3) and relative fluorescence intensities were determined using an LSR-II (Becton-Dickinson, San Diego, CA) and analyzed with FlowJo flow cytometry analysis software (Ashland, OR). Fluorescence minus one (FMO) controls were used to assist with gating.
Andemariam B., Adami A.J., McNamara J.T., Secor E.R., Guernsey L.A, & Thrall R.S. (2015). The sickle cell mouse lung: pro-inflammatory and primed for allergic inflammation. Translational research : the journal of laboratory and clinical medicine, 166(3), 254-268.
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4

Heparan Sulfate Expression Analysis

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Huh7.5.1 cells were seeded onto 12-wells plates and treated with siNT or SMAD6 siRNA as described above. After 72 h, cells were harvested and immunostained with a mouse anti-heparan sulfate delta antibody (3G10 epitope, US biological, 1:100), followed by incubation with a FITC-conjugated goat anti-mouse IgG antibody (Invitrogen, 1:1000). After extensively washing, cells were resuspended in cold PBS and subjected to flow cytometry on a FACS Canto flow cytometer (BD Biosciences) that collects ~40,000 gated events. FlowJo flow cytometry analysis software (Flowjo LLC) was used for analysis of HSPG expression levels under various treatment conditions.
Zhang F., Sodroski C., Cha H., Li Q, & Liang T.J. (2016). Infection of Hepatocytes with HCV Increases Cell Surface Levels of Heparan Sulfate Proteoglycans, Uptake of Cholesterol and Lipoprotein, and Virus Entry by Upregulating SMAD6 and SMAD7. Gastroenterology, 152(1), 257-270.e7.
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5

FACS analysis of Panc-1 cells treated with ODN-1 liposomes

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FACS analyses were carried out on Panc-1 cells (2.5 × 104) treated with 360 pmol oligonucleotide ODN-1 anchored to liposomes (96 ODN-1/liposome) for 1, 2 and 4 h. The cells were harvested at various time points after liposome transfection, washed with PBS and immediately analysed by FACSAria III flowcytometer (Becton-Dickinson, San Jose, CA, USA). A minimum of 104 cells for each sample were analyzed. The signal was detected by FL3 (680 nm) channel in log scale. The uptake data was obtained with FLOWJO flow cytometry analysis software (FLOWJO LLC, Ashland OR USA).
Cogoi S., Jakobsen U., Pedersen E.B., Vogel S, & Xodo L.E. (2016). Lipid-modified G4-decoy oligonucleotide anchored to nanoparticles: delivery and bioactivity in pancreatic cancer cells. Scientific Reports, 6, 38468.
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6

BrdU Labeling and Flow Cytometry Analysis of Tumor-Infiltrating Lymphocytes

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Tumor bearing BALB/c mice were injected with 2mg BrdU intraperitoneally in 200μl DPBS 24 hours before TIL isolation. Isolated TILs were stained using APC BrdU Flow Kit (BD Biosciences #51-9000019AK) adapting the manufacturer’s protocol. Briefly, cells were stained for surface antigens, then resuspended in BD Cytofix/Cytoperm buffer for 30 min on ice. Cells were washed with Perm/Wash and resuspended in BD Cytoperm Permeabilization Buffer Plus for 10 min on ice. Cells were then re-fixed/permeabilized overnight using the Foxp3/Transcription Factor Staining Buffer Set (eBioscience #00-5523-00). Cells were then treated with 30μg DNase for 1 hour at 37°C. Cells were then stained for intracellular proteins including BrdU for 30 min at room temperature. Data were acquired using the BD FACSCanto (BD Biosciences, San Jose, CA). Acquired data were analyzed using FlowJo Flow Cytometry Analysis Software (FlowJo LLC, Ashland, OR).
Vick S.C., Kolupaev O.V., Perou C.M, & Serody J.S. (2021). Anti-PD-1 checkpoint therapy can promote the function and survival of regulatory T cells.. Journal of immunology (Baltimore, Md. : 1950), 207(10), 2598-2607.
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7

Multiparametric Flow Cytometry of Murine Immune Cells

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Flow cytometry monoclonal antibodies against murine CD45 (30-F11 #11-0451-82), Foxp3 (FJK-16S #45-5773-82), PD-1 (J43 #48-9981-82), Ki67 (SolA15 #17-5698-80), Thy1.1 (HIS51 #45-0900-80), CTLA-4 (UC10-4B9 # 12-1522-82), and GITR (DTA-1 #25-5874-82) were purchased from Invitrogen. Monoclonal antibodies against murine CD4 (GK1.5 #100414), CD8 (53-6.7 #100722), PD1 (RMP1-30 #109103), LAP-TGFβ (TW7-16B4 #141405), CD25 (PC61 #102051), and BrdU (Bu20a #339808) were purchased from BioLegend. Monoclonal antibodies against murine Bcl-2 (3F11 #556537) were purchased from BD Biosciences, and monoclonal antibodies against murine Bim (C34C5 #948055) were purchased from Cell Signaling Technology. Cell viability was determined using Aqua Fluorescence Reactive Dye (Life Technologies #L34965). For flow cytometry, cells were surface stained, fixed/permeabilized overnight using the Foxp3/Transcription Factor Staining Buffer Set (eBioscience #00-5523-00), and intracellular staining performed the following day according to manufacturer’s instructions. Apoptosis was measured using PE Annexin V Apoptosis Detection Kit (BD Pharmingen #559763). Data were acquired using the BD FACSCanto or BD LSRFortessa (BD Biosciences, San Jose, CA). Acquired data were analyzed using FlowJo Flow Cytometry Analysis Software (FlowJo LLC, Ashland, OR).
Vick S.C., Kolupaev O.V., Perou C.M, & Serody J.S. (2021). Anti-PD-1 checkpoint therapy can promote the function and survival of regulatory T cells.. Journal of immunology (Baltimore, Md. : 1950), 207(10), 2598-2607.
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8

Lymphocyte subset analysis by flow cytometry

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The BD LSR II Flow cell cytometer at the Wisconsin Institute of Medical Research was used for sample analysis. FlowJo flow cytometry analysis software (Ashland, OR) was used to analyze the raw data. We initially gated for lymphocytes, then B cells & T cells, and finally the various subsets. Percentages of cells of a given phenotype were calculated as a function of the parent cell population. A representative figure demonstrating our gating strategy is seen in figure 1. These percentages were compared using analysis of variance (ANOVA) and the Fisher protected least significance difference (PLSD) test, with α = 0.05 (Statview 5.0.1, SAS, Cary, NC). Numerical results are presented as mean ± standard error of the mean.
Jonker M.A., Heneghan A.F., Fechner J.H., Pierre J.F., Sano Y., Lan J, & Kudsk K.A. (2015). Gut Lymphocyte Phenotype Changes after Parenteral Nutrition and Neuropeptide Administration. Annals of surgery, 262(1), 194-201.
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9

Mitochondrial Membrane Potential Assay

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MMP was determined by flow cytometry (FC 500 MPL; Beckman Coulter Inc., Fullerton, CA, USA) using 5,5ʹ,6,6ʹ-tetrachloro-1,1ʹ,3, 3ʹ-tetraethylbenzimidazol-carbocyanine iodide (JC-1) staining. JC-1 (C2006-1, Beyotime Institute of Biotechnology) is a lipophilic cationic dye that can selectively enter into mitochondria; its color reversibly changes with mitochondrial membrane depolarization. In normal cells with a high MMP, JC-1 spontaneously forms complexes (known as J-aggregates) and exhibits intense red fluorescence. In unhealthy cells with a low MMP, JC-1 remains in its monomeric form and exhibits green fluorescence.14 (link) HL-60 cells were harvested and incubated with JC-1 at 37°C for 20 minutes at 1 × 106 cells/mL. The cells were washed with JC-1 dye buffer (C2006-3, Beyotime Institute of Biotechnology) and analyzed using the FlowJo flow cytometry analysis software (FlowJo LLC, Ashland, OR, USA) at 525 nm excitation and 590 nm emission.
Xie D., Li Y.L., Wang G.F., Jiang J, & Sun L.R. (2021). Ultraviolet light-emitting diode irradiation induces reactive oxygen species production and mitochondrial membrane potential reduction in HL-60 cells. The Journal of International Medical Research, 49(5), 03000605211016623.
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10

Neutrophil Respiratory Burst Assay

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Neutrophil respiratory burst of each preparation was assessed using a neutrophil respiratory burst assay kit (601130, Sapphire Bioscience, New Zealand). Briefly, 2 aliquots of isolated neutrophils (~1 × 10 6 cells/mL) from all control animals were warmed (37°C) in 100 μL of assay buffer: RPMI 1640 cell culture medium (Life Technologies New Zealand Ltd.) supplemented with 10% fetal bovine serum of New Zealand origin (In Vitro Technologies) and 500 μL of 1 M calcium chloride. The dye, 10 μL of dihydrorhodamine 123 at 5 μg/mL, was added to both aliquots of cell suspension and incubated at 37°C for 15 min. Phorbol 12-myristate 13-acetate was added to 1 aliquot of cell suspension at a final concentration of 200 nM and incubated at 37°C for 45 min. After the incubation period, samples were centrifuged for 5 min at 500 × g, the supernatant discarded, and the cells resuspended in 300 μL of assay buffer and analyzed by flow cytometry for green fluorescence (~530 nm) using a BD FACSVerse (BD Biosciences New Zealand). Neutrophil populations were selected based on forward angle light scatter and side scatter, and fluorescence of unstimulated cells was compared with that of cells stimulated with phorbol 12-myristate 13-acetate using FlowJo flow cytometry analysis software version 10.4.2 (FlowJo, LLC, Ashland, OR).
Crookenden M.A., Phyn C.V.C., Turner S.A., Loor J.J., Smith A.I., Lopreiato V., Burke C.R., Heiser A, & Roche J.R. (2020). Feeding synthetic zeolite to transition dairy cows alters neutrophil gene expression. Journal of dairy science, 103(1).
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