Cellquest analysis software

Manufactured by BD

Sourced in United States, Germany

CellQuest is a flow cytometry data analysis software developed by BD. It provides users with tools to acquire, analyze, and manage flow cytometry data. The software enables users to display and interpret cytometric data through various visualization options, such as scatter plots and histograms.

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72 protocols using cellquest analysis software

Apoptosis Profiling by Flow Cytometry

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Annexin V/7-AAD Staining: Cells were stained by using Annexin V/7-AAD kit (Biolegend, California, USA). Briefly, 200 µL cell suspension was centrifuged and the supernatant was removed. The pellet was resuspended in 100 µL Annexin V binding buffer (Biolegend, San Diego, CA, USA) and 5 µL Annexin V-APC (BioLegend, San Diego, CA, USA) and 7-AAD (Biolegend, San Diego, CA, USA) were added, followed by incubation for 15 min at room temperature in the dark. Measurement was performed on the LSRII (Becton Dickinson, Franklin Lakes, NJ, USA) flow cytometer using CellQuest analysis software (Becton Dickinson, Franklin Lakes, NJ, USA).
To analyze caspase-3 cleavage, cells were washed and then resuspended in 200 μL 4% paraformaldehyde for 15 min at room temperature in the dark. Cells were permeabilized in methanol and incubated on ice for 30 min. For immunostaining, cells were incubated for 1 h with Cleaved Caspase-3 rabbit mAb -AF647 (Cell Signaling, Cambridge, UK). Measurement was performed on the LSRII flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA) using CellQuest analysis software (Becton Dickinson, Franklin Lakes, NJ, USA).

Pansy K., Feichtinger J., Ehall B., Uhl B., Sedej M., Roula D., Pursche B., Wolf A., Zoidl M., Steinbauer E., Gruber V., Greinix H.T., Prochazka K.T., Thallinger G.G., Heinemann A., Beham-Schmid C., Neumeister P., Wrodnigg T.M., Fechter K, & Deutsch A.J. (2019). The CXCR4–CXCL12-Axis Is of Prognostic Relevance in DLBCL and Its Antagonists Exert Pro-Apoptotic Effects In Vitro. International Journal of Molecular Sciences, 20(19), 4740.

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Cell Cycle and Apoptosis Analysis

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To profile cell cycle progression, we performed propidium iodine (PI) stain after doxorubicin treatments. One million cells were washed in phosphate‐buffered saline and then fixed in 70% ethanol at 4 °C for 45 min. The cells were pelleted and resuspended in 500 μL of PI staining solution composed of 50 μg mL⁻¹ PI and 0.1 mg mL⁻¹ RNase A on ice for 1 h in the dark. PI‐stained cells were analyzed by flow cytometry using a FACSCAlibur instrument with cellquest analysis software (BD Biosciences, San Jose, CA, USA). The extent of apoptosis was monitored by using the Annexin‐V FLUOS Staining Kit (Roche Applied Science, Indianapolis, IN, USA), according to the manufacturer's instructions. Briefly, cells were collected resuspended in 100 mL of Annexin‐V‐FLUOS labeling solution. After incubation at room temperature for 15 min in the dark, cells were analyzed by the flow cytometry using a FACSCAlibur instrument with cellquest analysis software (BD Biosciences).

Shih C., Chang Y., Chen Y., Ma C., Chen H., Yang C., Lu J., Tsai Y., Chen H, & Tan B.C. (2017). The PPARγ‐SETD8 axis constitutes an epigenetic, p53‐independent checkpoint on p21‐mediated cellular senescence. Aging Cell, 16(4), 797-813.

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Apoptotic DNA Fragmentation Analysis

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To analyze the fragmentation of genomic DNA in apoptotic cells, cells (1×10⁶ cells/ml) were incubated with reagents for 12 h. The cells were harvested, washed with PBS, and fixed using 1 ml of ice-cold 70% ethanol at 4℃ overnight. Fixed cells were labeled with 500µg/ml propidium iodide (PI) at 37℃ for 1 h. Genomic DNA was analyzed using a FACSCalibur flow cytometer (Becton Dickinson, San Jose, CA, USA) as previously described (26 (link),27 (link)). All data were calculated and displayed using the Cell Quest analysis software provided by Becton Dickinson.

Kim Y.C., Song S.B., Lee S.K., Park S.M, & Kim Y.S. (2014). The Nuclear Orphan Receptor NR4A1 is Involved in the Apoptotic Pathway Induced by LPS and Simvastatin in RAW 264.7 Macrophages. Immune Network, 14(2), 116-122.

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Nicotine-Induced Cell Cycle Arrest

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Cells were seeded in triplicate into 100-mm Petri dishes at a density of 1×10⁵ cells per plate and incubated overnight. The cells were then washed twice with PBS, and the media were changed to 0.5%-FBS-containing medium for 24 h. The RASMCs were exposed to nicotine at various concentrations (100 μM, 10 μM, 1 μM, and 0.1 μM) for different treatment periods (0, 24 h). The cells (1×10⁶) were harvested, washed twice with PBS, resuspended in 0.6 ml PBS, and fixed by the addition of 1.4 ml 100% ethanol at 4°C overnight. The fixed cells were rinsed twice with PBS, resuspended in propidium iodide (PI) solution containing 50 mg/ml PI and 50 mg/ml RNaseA (Sigma) in PBS, and incubated at 37°C for 30 min in the dark. The stained cells were analyzed using a FACScan Flow cytometer and Cell Quest analysis software (Becton Dickinson, San Jose, CA, USA). The flow cytometric analysis was repeated 3 times [17] (link).

He F., Li B., Zhao Z., Zhou Y., Hu G., Zou W., Hong W., Zou Y., Jiang C., Zhao D, & Ran P. (2014). The Pro-Proliferative Effects of Nicotine and Its Underlying Mechanism on Rat Airway Smooth Muscle Cells. PLoS ONE, 9(4), e93508.

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Evaluating THC Effects on Dendritic Cell Differentiation

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Human monocytes were prepared from PBMC by immunomagnetic depletion (Miltenyi Biotec, Auburn, CA) and T cells were purified using a combination of mAb (anti-CD14, anti-CD16, anti-CD19) and anti-mouse Ig-conjugated immunomagnetic beads (Dynal, Lake Success, NY). DC were differentiated from monocyte precursors by culturing adherent PBMC in X-VIVO 15 medium (Lonza; Walkersville, MD) supplemented with GM-CSF (800 U/ml) and IL-4 (100 to 500 U/ml) according to a standard protocol (Kiertscher and Roth 1996 (link)). The effects of THC on this differentiation process were assessed by adding THC (0.25 to 1.0 μg/ml) or diluent alone (containing ethanol/DMSO) 10 min before the addition of GM-CSF and IL-4. Dendritic cells were recovered from the flasks on day 7 and the expression of cell surface markers characterized by fluorescence-activated cell sorting (FACS) using a FACS Calibur^® cytometer with CellQuest^® analysis software (Becton Dickinson, San Jose, CA). For mixed leukocyte reactions (MLR) and cytokine assays, DC were further purified by depleting T cells, natural killer cells and B cells using lineage-specific mAb (anti-CD3, anti-CD19, anti-CD56) and immunomagnetic beads (Dynal).

Roth M.D., Castaneda J.T, & Kiertscher S.M. (2015). Exposure to Δ9-Tetrahydrocannabinol Impairs the Differentiation of Human Monocyte-derived Dendritic Cells and their Capacity for T cell Activation. Journal of neuroimmune pharmacology : the official journal of the Society on NeuroImmune Pharmacology, 10(2), 333-343.

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Annexin V-FITC Apoptosis Assay

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Apoptosis was detected with annexin V-FITC assay (BD Pharmingen, San Diego, CA, United States) according to manufacturer’s instructions. A375 cells were seeded in 35 mm culture dishes and allowed to attach overnight. The cells were treated with NAP-HBTA (100 μM) for 24-48-72 h. To detect early and late apoptosis, both adherent and floating cells were harvested together, washed twice with PBS and resuspended in annexin V binding buffer at a concentration of 10⁶ cells/mL. Subsequently, 5 μL of FITC-conjugatedAnnexin V and 5 μL of PI were added to 100 μL of the cell suspension (10⁵ cells). The cells were incubated for 20 min at room temperature in the dark and subsequently analyzed using a two-laser equipped FACSCalibur apparatus and the CellQuest analysis software (Becton Dickinson, Mountain View, CA, United States).

Ercolano G., De Cicco P., Frecentese F., Saccone I., Corvino A., Giordano F., Magli E., Fiorino F., Severino B., Calderone V., Citi V., Cirino G, & Ianaro A. (2019). Anti-metastatic Properties of Naproxen-HBTA in a Murine Model of Cutaneous Melanoma. Frontiers in Pharmacology, 10, 66.

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Cell Cycle Analysis by Flow Cytometry

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The cells (1 × 10⁶) were digested with a trypsin solution, then harvested and washed twice with cold PBS. The washed cells were rinsed twice with PBS, and re-suspended in a propidium iodine solution comprising containing 40 μg/mL propidium iodine and 100 lg/mL RNaseA (Sigma-Aldrich) in PBS without calcium and magnesium, then incubated at 37 °C for 30 min in the dark. The stained cells were passed through a nylon mesh sieve to remove cell clumps, and then analyzed with FACScan flow cytometer and the CELL QUEST analysis software (Becton Dickinson, San Jose, CA, USA). The flow cytometry analysis was repeated three times.

Gu Y., Zhang Z., Yin J., Ye J., Song Y., Liu H., Xiong Y., Lu M., Zheng G, & He Z. (2017). Epigenetic silencing of miR-493 increases the resistance to cisplatin in lung cancer by targeting tongue cancer resistance-related protein 1(TCRP1). Journal of Experimental & Clinical Cancer Research : CR, 36, 114.

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Mitochondrial Membrane Potential Assay

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MMP was determined by using the fluorescent dye DiOC₆. PC-3 and LNCaP cells were seeded at a density of 5×10⁴ cells/well in a 6-well culture dish and treated with different concentrations of DPT for 24 h. The cells were harvested and stained with 100 nM DiOC₆ at 37°C for 30 min. Cells were then analyzed by flow cytometry (FACSCalibur, Becton Dickinson, San Jose, CA, USA) with CellQuest analysis software (Becton Dickinson).

Kim S.H., Kim K.Y., Park S.G., Yu S.N., Kim Y.W., Nam H.W., An H.H., Kim Y.W, & Ahn S.C. (2017). Mitochondrial ROS activates ERK/autophagy pathway as a protected mechanism against deoxypodophyllotoxin-induced apoptosis. Oncotarget, 8(67), 111581-111596.

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HEK293 and MO59 Transduction Optimization

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HEK293 cells, seeded at 10⁵ cells per well of a six-well plate or 3 × 10⁵ cells per T25 flask 24 hours prior to transduction, were incubated with vector in the presence of 8 µg/ml of polybrene for 4 hours at 37°C. Transductions, performed in duplicate, utilized vector normalized for p24 (325 ng of p24 corresponding to an estimated MOI of five). GFP expression was assessed using a FACScan cytometer and CellQuest analysis software (Becton-Dickinson, San Jose, CA). For measurement of LUC expression, cell lysates were prepared from transduced cells, upon which 10 µg of protein per sample was added per well of a 96-well plate. LUC activity was measured as light intensity upon the addition of the LUC substrate, luciferin (Luciferase Assay System, Promega, Madison, WI).
MO59J and MO59K cells, seeded at 3 × 10⁵ cells per 100 mm dish 24 hours prior to transduction and transduced with vector supernatant containing 2000 ng of p24.

Shaw A.M., Joseph G.L., Jasti A.C., Sastry-Dent L., Witting S, & Cornetta K. (2016). Differences in Vector Genome Processing and Illegitimate Integration of Non-Integrating Lentiviral Vectors. Gene therapy, 24(1), 12-20.

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HEK293 and MO59 Transduction Optimization

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