Single cell analysis software

Manufactured by FlowJo

Sourced in United States

FlowJo Single Cell Analysis Software is a powerful tool for the analysis of flow cytometry data. It provides a comprehensive suite of features for the visualization, gating, and analysis of single-cell data. The software offers a user-friendly interface and advanced analytical capabilities to help researchers gain insights from their flow cytometry experiments.

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FlowJo v10.2 single cell analysis software FlowJo Single Cell Analysis Software v10 FlowJo X single cell analysis software FlowJo cell analysis software FlowJo software analysis

8 protocols using single cell analysis software

Comprehensive Immune Cell Profiling

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For immune population analysis, cells were stained in FACS buffer (PBS + 2% FCS + 1 mM EDTA) at 4°C for 20–30 min. Staining was performed using fluorochrome-conjugated antibodies for anti-CD45, -TCRb, -CD90.2, -CD19, -CD4, -CD8, -TCRgd, -NKp46, -CD11b, -CD11c, -F4/80, -I-A/E, -Ly6C (Biolegend, San Diego, CA); anti-Foxp3 (eBioscience, San Diego, CA). Fluorochromes were APC-Cy7, PE-Cy7, PE-texas red, pacific blue, Alexa fluor 700, FITC, PE and APC. Cells were washed twice with FACS buffer followed by centrifugation at 1500 rpm for 5 min and resuspension in the same buffer. To detect intracellular expression of Foxp3, cells were fixed and permeabilized using the Intracellular Fixation & Permeabilization buffer set (88–8824; eBioscience, San Diego, CA) according to the manufacturer’s protocol. Briefly, cells were resuspended in eBioscience Fix/Perm buffer for at least 30 min before washing and resuspended in Perm/Wash buffer containing antibody to Foxp3. Data was analyzed by Flowjo single cell analysis software (Flowjo LLC, Ashland, OR).

Singhal G., Fisher F.M., Chee M.J., Tan T.G., El Ouaamari A., Adams A.C., Najarian R., Kulkarni R.N., Benoist C., Flier J.S, & Maratos-Flier E. (2016). Fibroblast Growth Factor 21 (FGF21) Protects against High Fat Diet Induced Inflammation and Islet Hyperplasia in Pancreas. PLoS ONE, 11(2), e0148252.

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Multiparametric Flow Cytometry Immunophenotyping

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PBMC from human donors were stained with anti-human CD3 (UCHT1) PE and anti-human CD19 (SJ25-C1) APC-H7, murine spleen cells with anti-CD19 (eBio1D3) PE, anti-CD4 (RM4-5) APC, anti-CD11b (M1/70) eF450 and anti-CD11c (N418) PE/Cy7, murine peritoneal lavage cells with anti-CD11b (M1/70) eF450 and anti-F4/80 (BM8) PE, and murine epidermal cells with anti-CD49f (eBioGoH3 rat) PE antibodies at 4 °C for 30 min. Antibodies were purchased from Thermo Fisher Scientific Germany (Frankfurt a. M., Germany). Stained cells, or harvested cell culture cells, respectively, were washed with FACS buffer (Table 1) three times and resuspended in FACS buffer. Shortly before analysis, DAPI was added to a final concentration of 3 µM. Cells were sorted on a BD FACSAria™ III (Beckton Dickinson Germany, Heidelberg, Germany) excluding doublets and dead cells. Data were analyzed using FlowJo Single Cell Analysis Software (FLOWJO, LLC Data analysis software).

Schulz M.S., Sartorius von Bach C.B., Marinkovic E., Günther C., Behrendt R, & Roers A. (2023). Development of an RNase H2 Activity Assay for Clinical Screening. Journal of Clinical Medicine, 12(4), 1598.

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Annexin V and Sytox Apoptosis Assay

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Previous to flow cytometry analysis, cells were treated with 0.5 µM, 1.0 µM or 2.5 µM of either AzaC or AzadC for 72 h, harvested and washed twice with DPBS. Untreated cells served as a control. Afterward, cells were counted and 2 × 10⁵ cells per sample were transferred into a new tube. Apoptosis and necrosis were determined by using the FITC Annexin V Apoptosis Detection Kit (BioLegend 640,914) and SYTOX™ Red Dead Cell Stain (ThermoFisher S34859). To this end, cells were resuspended in 100 µL of Annexin V binding buffer supplemented with 1 µL of FITC-conjugated Annexin V, gently vortexed and incubated at RT for 15 min in the dark. Afterward, cells were put on ice and 100 µL of cell suspension (2 × 10⁵ cells) were filtered through a 35 µm strainer. 0.2 µL of Sytox was added just before the measurement. For the analysis, BD FACSCanto™ and FlowJo Single Cell Analysis Software (v10.8.0) were used. Gates were set once for the control sample and then applied to all other samples.

Aumer T., Gremmelmaier C.B., Runtsch L.S., Pforr J.C., Yeşiltaç G.N., Kaiser S, & Traube F.R. (2022). Comprehensive comparison between azacytidine and decitabine treatment in an acute myeloid leukemia cell line. Clinical Epigenetics, 14, 113.

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Measuring P-gp-mediated efflux in HeLa cells

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HeLa cells were trypsinized, counted, and 2 × 10⁵ cells per assay conditions were transferred into polystyrene flow cytometry tubes. Cells were incubated in Iscove's Modified Dulbecco's Medium (IMDM) containing either 1.5 μM rhodamine 123 (Rh123), 10 μM calcein-AM (CAM) or 10 μM JC-1 with or without P-gp modulators at 37°C for 45 mins. After incubation, cells were centrifuged, supernatant was removed, and resuspended in 200 μL of cold PBS and placed on ice to stop transporter activity. The efflux rate of P-gp and chimeras was determined by depletion of cellular ATP by incubation in PBS containing 20 mM 2-deoxy-d-glucose (2-DG) and 5 mM sodium azide for 20 mins. After incubation, 2-DG and sodium azide were removed and cells were incubated on ice in the presence of Rh123 for an additional 20 mins. Transport of Rh123 by P-gp was initiated when PBS containing 50 mM glucose was added to cells and the incubation temperature was adjusted to 37°C. A total of 10,000 individual cell-counting events were recorded using a FACSCanto II flow cytometer. A 488 nm blue laser was used to excite all fluorophores and emission was measured either at 530/30 nm (Rh123 and CAM) or 575/26 nm (JC-1). FACS data were analyzed using FlowJo Single Cell Analysis software (FlowJo, Ashland, OR).

Pluchino K.M., Hall M.D., Moen J.K., Chufan E.E., Fetsch P.A., Shukla S., Gill D.R., Hyde S.C., Xia D., Ambudkar S.V, & Gottesman M.M. (2016). Human-Mouse Chimeras With Normal Expression and Function Reveal That Major Domain Swapping is Tolerated by P-glycoprotein (ABCB1). Biochemistry, 55(7), 1010-1023.

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Quantifying Reactive Oxygen Species in Cells

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ROS release was determined in blood cells of infected mice by the formation of the fluorescent compound rhodamine-123 from dihydrorhodamine-123 (DHR-123, AAT Bioquest, California, USA). Staining was performed with 30 μl EDTA blood samples after erythrocyte lysis. Surface markers were stained as described above prior to ROS detection. Cells were incubated with 30 μg/ml DHR-123 in PBS for 20 min at 37°C in the dark. After washing with 4 ml of cold PBS 40 μl of cell suspensions were immediately analyzed using BD Accuri C6 (BD Biosciences, San José, USA) and FlowJo single cell analysis software (FlowJo LLC, Ashland, USA).

Papp S., Moderzynski K., Rauch J., Heine L., Kuehl S., Richardt U., Mueller H., Fleischer B, & Osterloh A. (2016). Liver Necrosis and Lethal Systemic Inflammation in a Murine Model of Rickettsia typhi Infection: Role of Neutrophils, Macrophages and NK Cells. PLoS Neglected Tropical Diseases, 10(8), e0004935.

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Comprehensive Immune Cell Profiling by Flow Cytometry

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To be able to exclude dead cells from the analysis, PBMCs were incubated with the viability dye Zombie-NIR (1:1000 in PBS, Biolegend, San Diego, USA) for 30 min in the dark. After washing in PBS, cells were stained with antibodies against CD3 (clone UCHT1; 1:50) and CD8 (clone RPA-T8; 1:500; both from Biolegend, San Diego, USA) in PBS/2% bovine serum albumin (BSA) for 20 min at 4 °C in the dark. Then, cells were washed in PBS/2% BSA and permeabilized with Fixation and Permeabilization buffer (BD Pharmingen, Heidelberg, Germany) for 20 min at 4 °C in the dark. The cells were washed twice in Perm/Wash buffer (BD Pharmingen, Heidelberg, Germany) and stained intracellularly with antibodies against CD4 (clone RPA-T4; 1:20), IFNγ (clone 4S.B3; 1:100), IL-17 (clone BL168; 1:20), IL-22 (clone 2G12A41; 1:20) and TNF (clone MAb11; 1:100; all from Biolegend, San Diego, USA) in Perm/Wash buffer for 30 min at 4 °C in the dark. Afterwards, the cells were washed again in Perm/Wash buffer and resuspended in PBS. Analyses were performed with a BD LSR II flow cytometer (BD Biosciences, San José, USA) and FlowJo single cell analysis software (FlowJo LLC, Ashland, USA).

Rauch J., Jochum J., Eisermann P., Gisbrecht J., Völker K., Hunstig F., Mehlhoop U., Muntau B, & Tappe D. (2022). Inflammatory cytokine profile and T cell responses in African tick bite fever patients. Medical Microbiology and Immunology, 211(2-3), 143-152.

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EdU-Based Proliferation Assay for MOLM-13 Cells

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To measure proliferation, EdU Flow Cytometry 488 kit (baseclick BCK-FC488-50) was used according to the manufacturer’s protocol. In brief, MOLM-13 (1 × 10⁶ per condition, 0.5 × 10⁶/mL) were treated with 0.5 µM, 1.0 µM or 2.5 µM of either AzaC or AzadC for 48 h, before EdU (final concentration 10 µM) was added for two additional hours. Untreated cells without EdU served as unstained control, and untreated cells with EdU served as untreated control. Afterward, the cells were harvested and washed with DPBS, including 1% (w/v) BSA, before fixation and permeabilization. Then, click chemistry was performed as described in the manual. Prior to flow cytometry measurement, the cells were filtered through a 35-µm strainer. For the analysis, BD FACSCanto™ and FlowJo Single Cell Analysis Software (v10.8.0) were used. Gates were set once for the control sample and then applied to all other samples.

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Cell Cycle Analysis by Flow Cytometry

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After 48 h of transfection, cells were collected by trypsinization and centrifugation at 300 g for 5 min. The supernatant media was discarded. Then, one ml of cold 70% ethanol was added and incubated at K20 8C for 1 h. Cells were centrifuged again at 300 g for 5 min and were washed twice with PBS. Cells were then subjected to propidium iodide (50 mg/ml in PBS), RNase (50 mg/ml) and Triton X-100 (0.1%) and incubated for 40 min at 37 8C. A total of 10 000 cells were examined by flow cytometry, with a MUSE cell analyser instrument (Merck Millipore) in triplicates. The results were analysed by FlowJo single-cell analysis software (FLOWJO, LLC, Ashland, OR, USA). Experiments including transfected cells, mock control and non-targeting control were performed in triplicate.

Salajegheh A., Vosgha H., Md Rahman A., Amin M., Smith R.A, & Lam A.K. (2015). Modulatory role of miR-205 in angiogenesis and progression of thyroid cancer. Journal of molecular endocrinology, 55(3).

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