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Flowjox v10

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FlowJoX v10.0.7 is a software application designed for the analysis and visualization of flow cytometry data. It provides tools for data management, gating, and statistical analysis of multi-dimensional flow cytometry datasets.

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Facscanto 2, by BD (3 mentions) Trustain fcx, by BioLegend (3 mentions) Facsuite v 1, by BD (2 mentions) Compbead, by BD (2 mentions) Anti mouse igg pe, by Jackson ImmunoResearch (2 mentions) Facsverse, by BD (1 mentions) Facsaria 3, by BD (1 mentions) Lsrii fortessa, by BD (1 mentions) Foxp3 staining buffer set, by Thermo Fisher Scientific (1 mentions) Intracellular fixation and permeabilization buffer set, by Thermo Fisher Scientific (1 mentions) Propidium iodide, by Merck Group (1 mentions) Facsverse flow cytometer, by BD (1 mentions) Cytometer setup tracking module, by BD (1 mentions) Facsaria 3 sorter, by BD (1 mentions) Pan b cell isolation kit, by Miltenyi Biotec (1 mentions) Lsrfortessa, by BD (1 mentions) Lsr 2, by BD (1 mentions)

10 protocols using flowjox v10

1

Extracellular Vesicle Staining and Quantification

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Extracellular vesicles (EVs) represent extracellular shuttles capable of transporting nucleic acid molecules (DNA and RNA) or protein outside the cell. For the EV staining, a method recently optimized in our laboratories was applied [60 (link),61 ]. In detail, EVs were stained by adding 0.5 µL of FITC-conjugated phalloidin and lipophilic cationic dye-LCD (BD Biosciences—Catalogue, #626267, Custom Kit), to 100 µL of supernatants. After 45 min of staining (RT, in the dark), 200 mL of PBS 1× were added to each tube and 1 × 106 events/sample were acquired by flow cytometer (FACSVerse, BD Biosciences). Parameters were optimized as already described [60 (link),61 ]. All the state-of-the-art recommendations were taken into consideration.
The evaluation of non-specific fluorescence was obtained by acquiring fluorescence minus one (FMO) and the isotype controls. Compensation was assessed using comp beads (BD Biosciences) and single stained fluorescent sample [62 (link)]. Data were analyzed using FACSuite v 1.0.6.5230 (BD Biosciences) and FlowJo X v 10.0.7 (Tree Star, Ashland, OR, USA) software. EV concentrations were obtained by volumetric count [60 (link),61 ,63 (link),64 (link)].
Avolio F., Martinotti S., Khavinson V.K., Esposito J.E., Giambuzzi G., Marino A., Mironova E., Pulcini R., Robuffo I., Bologna G., Simeone P., Lanuti P., Guarnieri S., Trofimova S., Procopio A.D, & Toniato E. (2022). Peptides Regulating Proliferative Activity and Inflammatory Pathways in the Monocyte/Macrophage THP-1 Cell Line. International Journal of Molecular Sciences, 23(7), 3607.
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2

SARS-CoV-2 RBD-ACE2 Binding Inhibition Assay

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Percentage of inhibition of RBD binding to ACE2 by plasma/serum was analyzed through a flow cytometry cell-based assay as previously described (25 (link)). This assay was validated by direct comparison of 50% inhibitory concentration (IC50) neutralization values obtained applying a pseudovirus-based neutralization assay using HIV-based pseudovirus and ACE2 expressing 293T cells (56 (link)). Briefly, a murine stable cell line expressing the human ACE2 receptor (1.2 × 103 300.19-ACE2 cells per well in a 96-well plate) was incubated with RBD-mFc fusion proteins (4 μg/mL), previously exposed to plasma/serum samples at a dilution of 1:50 in PBS for 30 min at 4°C. Cells were stained with anti-mouse IgG-PE (Jackson ImmunoResearch), washed, and analyzed by Flow cytometry using standard procedures. Samples were acquired with a FACSCanto II (BD Biosciences) and analyzed with FlowJo Xv10.0.7 (Tree Star, Inc) software (57 (link)). The surrogate neutralization assay was performed in a subset of 50 and 265 samples from the Munich and Barcelona cohorts, respectively. Samples were randomly selected covering the whole range of IgG-RBD levels at the Luminex.
Aguilar R., Li X., Crowell C.S., Burrell T., Vidal M., Rubio R., Jiménez A., Hernández-Luis P., Hofmann D., Mijočević H., Jeske S., Christa C., D'Ippolito E., Lingor P., Knolle P.A., Roggendorf H., Priller A., Yazici S., Carolis C., Mayor A., Schreiner P., Poppert H., Beyer H., Schambeck S.E., Izquierdo L., Tortajada M., Angulo A., Soutschek E., Engel P., Garcia-Basteiro A., Busch D.H., Moncunill G., Protzer U., Dobaño C, & Gerhard M. (2023). RBD-Based ELISA and Luminex Predict Anti-SARS-CoV-2 Surrogate-Neutralizing Activity in Two Longitudinal Cohorts of German and Spanish Health Care Workers. Microbiology Spectrum, 11(1), e03165-22.
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3

SARS-CoV-2 Neutralization Assay using Flow Cytometry

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Percentage of inhibition of RBD binding to ACE2 by plasma/serum was analyzed through a flow cytometry cell-based assay as previously described (25 (link)). This assay was validated by direct comparison of 50% inhibitory concentration (IC50) neutralization values obtained applying a pseudovirus-based neutralization assay using HIV-based pseudovirus and ACE2 expressing 293T cells (56 (link)). Briefly, a murine stable cell line expressing the human ACE2 receptor (1.2 × 103 300.19-ACE2 cells per well in a 96-well plate) was incubated with RBD-mFc fusion proteins (4 μg/mL), previously exposed to plasma/serum samples at a dilution of 1:50 in PBS for 30 min at 4°C. Cells were stained with anti-mouse IgG-PE (Jackson ImmunoResearch), washed, and analyzed by Flow cytometry using standard procedures. Samples were acquired with a FACSCanto II (BD Biosciences) and analyzed with FlowJo Xv10.0.7 (Tree Star, Inc) software (57 (link)). The surrogate neutralization assay was performed in a subset of 50 and 265 samples from the Munich and Barcelona cohorts, respectively. Samples were randomly selected covering the whole range of IgG-RBD levels at the Luminex.
Aguilar R., Li X., Crowell C.S., Burrell T., Vidal M., Rubio R., Jiménez A., Hernández-Luis P., Hofmann D., Mijočević H., Jeske S., Christa C., D'Ippolito E., Lingor P., Knolle P.A., Roggendorf H., Priller A., Yazici S., Carolis C., Mayor A., Schreiner P., Poppert H., Beyer H., Schambeck S.E., Izquierdo L., Tortajada M., Angulo A., Soutschek E., Engel P., Garcia-Basteiro A., Busch D.H., Moncunill G., Protzer U., Dobaño C, & Gerhard M. (2023). RBD-Based ELISA and Luminex Predict Anti-SARS-CoV-2 Surrogate-Neutralizing Activity in Two Longitudinal Cohorts of German and Spanish Health Care Workers. Microbiology Spectrum, 11(1), e03165-22.
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4

Characterization of T Cell Subsets

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2 × 106 single cell suspensions prepared from the lung or spleen were resuspended in 200 ul of PBS with 2% fetal bovine serum and blocked with TruStain fcX (BioLegend, San Diego, CA). T cell subsets were identified with antibodies against CD45 (clone 30-F11, BV510), TCRβ (clone H57–597, PerCP-Cy5.5), CD8 (clone 53–6.7, BV785), CD44 (clone IM7, APC-eFluor780), CD62L (clone MEL-14, FITC), CD127 (clone 678 A7R34, PE-Dazzle594), and KLRG1 (clone 2F1/KLRG1, PE-Cy7). All antibodies were purchased from BioLegend, eBioscence (Thermo Fisher Scientific), or BD Biosciences (San Jose, CA). H-2K(b)-p79 or H-2D(b)-p56 MHC-peptide complex was provided as dextramers conjugated to PE or APC (Immundex, Fairfax, VA). The data was collected on a CytoFLEX flow cytometer (Beckman Coulter) and analyzed using FlowJoX v10.0.7 (Treestar Inc., Ashland, OR). Cells were first gated as live per exclusion of Alexa Fluor™ 700 NHS Ester uptake and singlet lymphocytes based on forward and side scatter parameters prior to subgating.
Bland W.A., Owens S., McEvoy K., Hogan C.H., Boccuzzi L., Kirillov V., Khairallah C., Sheridan B.S., Forrest J.C, & Krug L.T. (2023). Replication-dead gammaherpesvirus vaccine protects against acute replication, reactivation from latency, and lethal challenge in mice. bioRxiv.
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5

Flow Cytometry Immunological Protocol

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Flow cytometry was performed according to the Guidelines for the use of flow cytometry and cell sorting in immunological studies (Cossarizza et al., 2019 (link)). To minimize non-specific staining, all incubations were carried out in the presence of 20% rabbit serum (Linus) and 1% of fetal bovine serum in PBS. Adherent cells were harvested by incubation with 2 mM EDTA or when indicated by treatment with trypsin at 37°C. For intracellular staining, cells were fixed and permeabilized with either the Intracellular fixation and permeabilization buffer set or Foxp3 staining buffer set (eBioscience, Thermo Fisher Scientific) following manufacturer's instructions, before antibody incubations. Prior to cytometry analysis cell suspensions from spleens, lungs, salivary glands, and salivary gland draining lymph nodes were washed and filtered through a 70-μm-cell strainer (Biologix). All samples were acquired with LSRII Fortessa, FACSAria III or FACSCanto II flow cytometers (BD Biosciences) and analyzed with FlowJo Xv10.0.7 (Tree Star, Inc) software. At least two independent experiments were carried out for each subject of analysis.
Angulo G., Zeleznjak J., Martínez-Vicente P., Puñet-Ortiz J., Hengel H., Messerle M., Oxenius A., Jonjic S., Krmpotić A., Engel P, & Angulo A. (2021). Cytomegalovirus restricts ICOSL expression on antigen-presenting cells disabling T cell co-stimulation and contributing to immune evasion. eLife, 10, e59350.
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6

Cell Cycle and Apoptosis Analysis

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Cell cycle and apoptosis were analyzed as described [16 (link)]. Briefly, MDA-MB-231 cells were incubated in 6-well plates at a density of 2.25 × 105 cells/well for 24 h. Cells were then treated with hydroquinone 10 or DMSO only for 48 h. For cell cycle analysis, cells were stained with propidium iodide (PI; Sigma-Aldrich, Sintra, Portugal); cell cycle scattering was analyzed by flow cytometry and cell cycle phases were quantified using FlowJoX v10.0.7 (Treestar, Woodburn, OR, USA).
Brandão P., Moreira J., Almeida J., Nazareth N., Sampaio-Dias I.E., Vasconcelos V., Martins R., Leão P., Pinto M., Saraíva L, & Cidade H. (2020). Norhierridin B, a New Hierridin B-Based Hydroquinone with Improved Antiproliferative Activity. Molecules, 25(7), 1578.
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7

Tear Extracellular Vesicle Characterization

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Tear samples were analysed as previously reported16 (link),19 (link),20 (link),35 (link). Briefly, 100 μL of tears were stained using a reagent mix, as detailed in Table S1. Samples were then incubated for 45 min, at room temperature, in the dark. After adding 200 μL of PBS 1X to each tube, 1 × 106 events/sample were acquired by a FACSVerse flow cytometer (BD Biosciences). The trigger threshold was set on the allophycocyanin (APC) channel, which is the channel in which the LCD (a pan EV marker) emits (threshold placed at 200/262,144)20 (link),36 (link). For all used parameters the height (H) signals and the bi-exponential/logarithmic modes were selected. Current guidelines for flow cytometry analyses and for EV studies were taken into account37 (link),38 (link). The Cytometer Setup & Tracking Module (BD Biosciences) was used for the daily quality check. Compensation was assessed using CompBeads (BD Biosciences) and single stained fluorescent samples. Data were analysed using FACSuite v 1.0.6.5230 (BD Biosciences) and FlowJo X v 10.0.7 (TreeStar, Ashland, OR, USA) software. EVs concentrations were obtained by the volumetric count function. With the used dilution no swarm effects occurred.
Pieragostino D., Lanzini M., Cicalini I., Cufaro M.C., Damiani V., Mastropasqua L., De Laurenzi V., Nubile M., Lanuti P., Bologna G., Agnifili L, & Del Boccio P. (2022). Tear proteomics reveals the molecular basis of the efficacy of human recombinant nerve growth factor treatment for Neurotrophic Keratopathy. Scientific Reports, 12, 1229.
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8

Characterization of T Cell Subsets

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2 × 106 single cell suspensions prepared from the lung or spleen were resuspended in 200 ul of PBS with 2% fetal bovine serum and blocked with TruStain fcX (BioLegend, San Diego, CA). T cell subsets were identified with antibodies against CD45 (clone 30-F11, BV510), TCRβ (clone H57–597, PerCP-Cy5.5), CD8 (clone 53–6.7, BV785), CD44 (clone IM7, APC-eFluor780), CD62L (clone MEL-14, FITC), CD127 (clone 678 A7R34, PE-Dazzle594), and KLRG1 (clone 2F1/KLRG1, PE-Cy7). All antibodies were purchased from BioLegend, eBioscence (Thermo Fisher Scientific), or BD Biosciences (San Jose, CA). H-2K(b)-p79 or H-2D(b)-p56 MHC-peptide complex was provided as dextramers conjugated to PE or APC (Immundex, Fairfax, VA). The data was collected on a CytoFLEX flow cytometer (Beckman Coulter) and analyzed using FlowJoX v10.0.7 (Treestar Inc., Ashland, OR). Cells were first gated as live per exclusion of Alexa Fluor™ 700 NHS Ester uptake and singlet lymphocytes based on forward and side scatter parameters prior to subgating.
Bland W.A., Owens S., McEvoy K., Hogan C.H., Boccuzzi L., Kirillov V., Khairallah C., Sheridan B.S., Forrest J.C, & Krug L.T. (2023). Replication-dead gammaherpesvirus vaccine protects against acute replication, reactivation from latency, and lethal challenge in mice. bioRxiv.
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9

Detailed Flow Cytometry of Murine B Cell Subsets

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For the analysis of B cells, 2 × 106 splenocytes were resuspended in 200 µl of fluorescence-activated cell sorter (FACS) buffer (PBS with 2% fetal bovine serum) and blocked with TruStain fcX (clone 93; BioLegend, San Diego, CA). The cells were washed and stained to identify B cell subsets with fluorophore-conjugated antibodies against CD19 (clone 6D5), B220 (clone RA3-682) CD138 (clone 281-2), CD95 (clone 15A7), and IgD (clone 11-26c.2a) or biotinylated antibodies against GL7 (clone GL-7), IgG1 (clone RMG1-1), IgG2b (clone R12-3), IgG2c (clone RMG2a-62), or IgG3 (clone R40-82) that were detected by the use of secondary streptavidin-conjugated allophycocyanin. T cell subsets were identified with antibodies against CD4 (clone GK1.5), CD8 (clone 53-6.7), Vβ4 (clone KT4), CD44 (clone IM7), and CD62L (clone MEL-14). For sorting, splenocytes were first enriched for B cells by depletion of non-B cells with magnetic microbeads (Pan B cell isolation kit; Miltenyi Biotec, Auburn, CA) and then sorted using CD19 conjugated to phycoerythrin and a FACSAria III sorter (BD Biosciences). All antibodies were purchased from BioLegend or BD Biosciences (San Jose, CA). The data were collected using a Dxp-8 FACScan flow cytometer (Cytek Development, Fremont, CA) and analyzed using FlowJoX v10.0.7 (Treestar Inc., Ashland, OR).
Reddy S.S., Foreman H.C., Sioux T.O., Park G.H., Poli V., Reich N.C, & Krug L.T. (2016). Ablation of STAT3 in the B Cell Compartment Restricts Gammaherpesvirus Latency In Vivo. mBio, 7(4), e00723-16.
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10

Colonic Tissue Leukocyte Profiling

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Isolated lamina propria cells were incubated with Fc block, stained with antibodies and flow cytometry was performed on a BD LSRII or BD LSRFortessa. Data were analyzed on FlowJo X v.10.0.7 (Tree Star). See Supplemental Figure 1 for gating strategy. Briefly, immune cell subsets were identified among live CD45+ cells after doublet exclusion. For determination of colonic tissue or colonic vascular localization of lamina propria leukocytes, 5µl of anti-CD45 APC in 200µl PBS was injected intravenously (i.v.) 5 minutes before euthanasia. This method allows for the differential identification of cells that are extravascular (at least excluded from antibody labeling) and likely located within the colonic tissue versus cells that are within the colonic vascular lumen. Total cell subsets were determined by multiplying the percentage of the cell subset, as determined by flow cytometry, by the total cell counts.
Frasch S.C., McNamee E.N., Kominsky D., Jedlicka P., Jakubzick C., Berry K.Z., Mack M., Furuta G.T., Lee J.J., Henson P.M., Colgan S.P, & Bratton D.L. (2016). G2A signaling dampens colitic inflammation via production of IFNγ. Journal of immunology (Baltimore, Md. : 1950), 197(4), 1425-1434.
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