Automated 16-bit fluorescent image acquisition was performed using MetaMorph 7.8 (Molecular Devices) and the MAC5000 controller (Ludl). The multiparameter images of the section of the cardiac lobe were combined into an image stack (*.stk file). The variable-sized image stack contained positional metadata that allowed subsequent reconstruction into an image montage. The image stack was processed using standard MetaMorph filters and segmented into cell nuclei and cytoplasmic features using custom routines created using CellProfiler (Broad Institute, Cambridge, MA, USA). The segmented images from CellProfiler were reconstructed into image montages of the cardiac lobe using FCS Express 5 software (De Novo Software, Los Angeles, CA, USA). Using FCS Express 5 (De Novo), the high expressing SMA cells on the dot plot were gated and the corresponding cells in the image montage were highlighted.
Fcs express 5
Manufactured by De Novo Software
Sourced in United States
FCS Express 5 is a software application designed for the analysis and visualization of flow cytometry data. It provides a comprehensive suite of tools for processing, gating, and reporting flow cytometry experiments.
Automatically generated - may contain errors
Lab products found in correlation
Facscanto 2, by BD (8 mentions)
Accuri c6 flow cytometer, by BD (5 mentions)
Facscalibur flow cytometer, by BD (5 mentions)
Facsdiva software, by BD (5 mentions)
Flow cytometry, by Sysmex (4 mentions)
Accuri c6, by BD (4 mentions)
Cyan adp, by Beckman Coulter (4 mentions)
Facscalibur, by BD (4 mentions)
Propidium iodide, by Merck Group (4 mentions)
Ionomycin, by Merck Group (3 mentions)
Golgistop, by BD (3 mentions)
Annexin 5 fitc apoptosis detection kit, by Keygen Biotech (3 mentions)
Trypsin edta, by Thermo Fisher Scientific (3 mentions)
Mitosox red, by Thermo Fisher Scientific (2 mentions)
Ma 900 multi application sorter, by Sony (2 mentions)
Ultracomp ebeads plus, by Thermo Fisher Scientific (2 mentions)
Facs canto 2 flow cytometer, by De Novo Software (2 mentions)
Tim 3, by Thermo Fisher Scientific (2 mentions)
Lag 3, by Thermo Fisher Scientific (2 mentions)
Ique screener plus flow cytometry analyzer, by Intellicyt (2 mentions)
94 protocols using fcs express 5
1
Automated Fluorescent Imaging of Cardiac Lobe
2
Flow Cytometry Analysis of PAM Markers
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PAMs were blocked with 1% BSA-PBS for 30 min at 37°C. Then the cells were incubated with antibodies against CD163 (mouse anti-human CD163 antibody, AbD Serotec, MCA1853) or CD169 (FITC-conjugated mouse anti-pig CD169 antibody, BIO-RAD, MCA2316F) at 37°C for 1 h. After washing with PBS three times, PAMs for detecting CD163 were incubated with Alexa Fluor 488-labeled goat anti-mouse IgG antibody (Invitrogen) at 37°C for 1 h. After washing with PBS three times, PAMs were immediately analyzed on a BD LSRFortessa flow cytometer (BD Biosciences) with FCS Express 5 software (De Novo Software). A minimum of 10,000 cells were analyzed for each sample.
3
Multiparametric Flow Cytometry Analysis
4
Mitochondrial ROS Production Analysis
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Production of mitochondrial reactive oxygen species (ROS) was assessed by flow cytometry as the O2•− production after 2.5 h of incubation using MitoSOX™ Red (Thermo Fisher Scientific, Waltham, MA, USA), following a previously published protocol [35 (link),36 (link)]. Prior to incubation, cells were labelled with 2.5 µM MitoSOX in Locke’s solution, and then incubated at 37 °C for 30 min, washed, and analysed in an AccurTM C6 flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA). The FCS Express 5 software (De Novo Software, Pasadena, CA, USA) was used to analyse the events recorded (1 × 104 Cells).
5
Annexin V-FITC and PI Staining of HEK 293 Cells
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HEK 293 cells were grown on 100-mm Petri dishes. About 106 cells were harvested and resuspended in Annexin V binding buffer. Then cells were incubated with Annexin V-FITC (BD Biosciences) and propidium iodide (PI) (Biolegend) for 15 min in the dark at room temperature. Cells were washed and fixed with 3.7% formaldehyde solution for 10 min and subjected to a flow cytometric analysis using FACScan flow cytometer (Becton Dickinson). Results were analyzed with FCS Express 5 software (De Novo Software).
6
PLS-488 Uptake in Raw 264.7 Cells
7
Live/Dead Assessment of Bacterial Cells
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Live/dead assessment
of the TiO2 bulk- and TiO2 nanoparticle-treated
bacterial cells was done as per the protocol by Jung et al.28 (link) The treated bacterial culture was washed with
PBS and incubated with PI (30 μM) and Syto 9 (20 μM) stains
for 15 min at room temperature. The red fluorescence of PI was collected
in the BL3 filter (647/10 band pass) of the cytometer, whereas the
green fluorescence of Syto 9 was collected in the BL1 filter (530/30).
The data were processed in FCS Express 5 software (Denovo, Los Angeles,
CA), and the dead/live ratio was calculated and plotted as a graph
by Graphpad Prism 5.
of the TiO2 bulk- and TiO2 nanoparticle-treated
bacterial cells was done as per the protocol by Jung et al.28 (link) The treated bacterial culture was washed with
PBS and incubated with PI (30 μM) and Syto 9 (20 μM) stains
for 15 min at room temperature. The red fluorescence of PI was collected
in the BL3 filter (647/10 band pass) of the cytometer, whereas the
green fluorescence of Syto 9 was collected in the BL1 filter (530/30).
The data were processed in FCS Express 5 software (Denovo, Los Angeles,
CA), and the dead/live ratio was calculated and plotted as a graph
by Graphpad Prism 5.
8
Multiparametric Flow Cytometry Analysis
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Cell surface and intracellular staining were performed using antibodies including CD3 (eFluor 450; eBioscience, Waltham, MA, United States; clone UCHT1), CD8a (FITC, eBioscience, clone RPA-T8; or APC-eFluor 780, eBioscience, clone RPA-T8), TRBV4-1 (PE; Miltenyi Biotec, Bergisch Gladbach, Germany; clone REA871), Ghost Dye (Violet 510; Tonbo Biosciences, San Diego, CA, United States), IFN-γ (PE-Cyanine7, eBioscience, clone 4S.B3), T-bet (BV421; BD Biosciences, San Jose, CA, United States; clone O4-46), Granzyme A (PE-Cyanine7, eBioscience, clone CB9), Granzyme B (FITC, BD eBioscience, clone GB11), Granzyme K (eFluor 660, eBioscience, clone G3H69), PRF1 (BV421, BD Biosciences, clone δG9), CCL4 (PerCP-eFluor 710, eBioscience, clone FL34Z3L), CCL5 (eFluor 660, eBioscience, clone VL1), and CXCR3 (PE-Cyanine7, eBioscience, clone CEW33D). Intracellular staining was performed on T cells stimulated with PMA (50 ng/mL; Sigma-Aldrich, St. Louis, MO, United States) and ionomycin (1 mM; Sigma-Aldrich) in the presence of GolgiStop (2/3 μL/mL, BD Biosciences) and Grid-plug (1 μL/mL, BD Biosciences) for 5 h. Flow cytometry was performed on a BD FACSCanto II flow cytometer using BD FACSDiva Software and FCS Express 5 software (De Novo Software, Los Angeles, CA, United States).
9
MPE Tumor Cell and Macrophage Isolation Protocol
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The antibodies for flow cytometry, including anti-CD11b, anti-F4/80, anti-CD206, and anti-MHC-II monoclonal antibodies, were purchased from Invitrogen (Carlsbad, CA, USA) and BD Biosciences (San Jose, CA, USA). All experiments were analyzed by flow cytometry on a FACS Canto II system (BD Biosciences), and data were examined using the FCS Express 5 software program (De Novo Software, Los Angeles, CA, USA). The flow cytometry methods and strategic steps taken are stated in detail in our previous publications [14 (link), 23 (link)]. Tumor cells in MPE (MC38-GFP) can be screened out by flow cytometry. MPE macrophages were isolated using anti-F4/80 microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany). The purity of macrophages was >90%, which was confirmed by flow cytometry, and cells were cultured in DMEM with 10% FBS for further use.
10
Plasma EV Profiling via Flow Cytometry
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EVs from 20 µl plasma were utilized for flow cytometry-based profiling. EV pellets were resuspended in double-filtered PBS (df-PBS) by 100 nm filters (MilliporeSigma) and stained with fluorescence-conjugated antibodies against human fibrinogen alpha chain (FGA), fibrinogen beta chain (FGB), and fibrinogen gamma chain (FGG) (Novus Biologicals). The high-resolution Sony MA 900 Multi-Application Sorter (Sony Biotechnology) was configured to ensure acquisition events of df-PBS <10 events/second; relative size distribution of EVs were estimated using reference beads with mean size 100, 1000 and 6000 nm (Bangs Laboratories) (6 (link)). The fluorescence background was determined using unstained and antibody-stained EVs and UltraComp™ eBeads Plus (ThermoFisher Scientific). The percentages (%) of plasma EVs carrying each surface marker were determined using flow cytometry; flow cytometric data analysis was performed using FCS Express 5 software (De Novo Software).
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