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Facsdiva software package

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The FACSDiva software package is a comprehensive flow cytometry data analysis and management solution. It provides tools for data acquisition, compensation, gating, and visualization. The software is designed to simplify the analysis of complex flow cytometry data.

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Lab products found in correlation

Lsrii flow cytometer, by BD (10 mentions) Pd 1 bv421 clone eh12.2h7, by BioLegend (3 mentions) Cd95 bv605 clone dx2, by BioLegend (3 mentions) Cd28 pe cy5.5 clone cd28 2, by Beckman Coulter (3 mentions) Cd62l pe clone sk11, by BD (3 mentions) Facscanto 2 flow cytometer, by BD (3 mentions) Anti cd3 and anti cd28 antibodies mouse anti human, by BD (2 mentions) Cd8 bv711 clone rpa t8, by BioLegend (2 mentions) Cd28 ecd clone cd28 2, by Beckman Coulter (2 mentions) Foxp3 permeabilization kit, by BD (2 mentions) Software package, by FlowJo (2 mentions) Cd45ra pe cy7 clone 5h9, by BD (2 mentions) Golgistop protein transport inhibitor, by BD (2 mentions) Ifn γ pe clone b7, by BD (2 mentions) Tnf α af700 clone mab11, by BD (2 mentions) Il 2 bv605 clone mq1 17h12, by BD (2 mentions) Carboxyfluorescein succinimidyl ester (cfse), by Thermo Fisher Scientific (1 mentions) Annexin 5 fitc apoptosis detection kit, by Merck Group (1 mentions) Lsr 2 flow cytometer, by BD (1 mentions) Cd4 bv650 clone okt4, by BioLegend (1 mentions)

16 protocols using facsdiva software package

1

CFSE-Based PBMC Proliferation Assay

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To evaluate cell proliferation, 1 × 107 PBMC in 10 ml of PBS were labeled with 5,6-carboxyfluorescein succinimidyl ester (CFSE, 0.5 μM, Invitrogen, Eugene, USA) according to the manufacturer's instructions. 2 × 106 cells/well were seeded in 6 well microtiter plates (TPP, Trasadingen, Switzerland) in 5 ml of 786-0VHL− or 786-0VHL+ conditioned media or in co-culture with 3 × 105 786-0VHL− / 786-0VHL+ cells/well in the presence of conditioned media followed by direct stimulation with plate-bound anti-CD3 and anti-CD28 antibodies (mouse anti-human, 1 μg/ml each, BD Biosciences) and maintained for 5 days in culture. 10,000 events were analyzed on a BD FACSCanto II flow cytometer in combination with the FACSDiva software package (BD Biosciences) in a time kinetic. The results were expressed as percentage of proliferating cells.
Proliferation assays in the presence of MnSOD2 were analyzed as described above in the absence and presence of 1U/ml recombinant MnSOD2 (Abnova; Pforzheim, Germany) within the culture medium for up to 40 hours.
Stehle F., Leisz S., Schulz K., Schwurack N., Weber N., Massa C., Kalich J., Fahldieck C, & Seliger B. (2015). VHL-dependent alterations in the secretome of renal cell carcinoma: Association with immune cell response?. Oncotarget, 6(41), 43420-43437.
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2

Apoptosis Induction by Salinomycin and DCA

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Cells were seeded in a 6-well plate at a density of 300,000 (HCT116) and 400,000 (DLD-1) cells per well. After 24 h, cells were treated with salinomycin, DCA, or drug combination at doses selected during the cell viability assay experiment: 0.25 μM of salinomycin, 15 mM of DCA, and the combination of two. After 48 h, cells were collected and washed by centrifugation. For apoptosis detection, we used an Annexin V-FITC apoptosis detection kit (Sigma Aldrich, USA). Cells were resuspended in 500 μL of binding buffer; subsequently, 5-μL annexin V conjugated with FITC and 5-μL propidium iodide (PI) solution (100 μg/mL) were added to the mixture, and cells were maintained at room temperature in the dark for 5 min. Cells were analyzed for apoptosis by a BD LSR II flow cytometer (BD Biosciences, USA) and an FACS Diva software package (BD Biosciences, USA).
Skeberdytė A., Sarapinienė I., Aleksander-Krasko J., Stankevičius V., Sužiedėlis K, & Jarmalaitė S. (2018). Dichloroacetate and Salinomycin Exert a Synergistic Cytotoxic Effect in Colorectal Cancer Cell Lines. Scientific Reports, 8, 17744.
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3

Multicolor Flow Cytometry Analysis

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Multicolor flow cytometric analysis was performed on whole blood or a cell suspension using predetermined optimal concentrations of the following fluorescently conjugated monoclonal antibodies (MAbs): CD3-APC-Cy7 (clone SP34-2), CD95-PE-Cy5 (clone DX2), Ki-67-AF700 (clone B56), HLA-DR-PerCP-Cy5.5 (clone G46-6), CCR7-FITC (clone 150503), CCR5-APC (clone 3A9), and CD45RA-PECy7 (clone L48) from BD Biosciences; CD8-BV711 (clone RPA-T8), CD4-BV650 (clone OKT4), and PD-1-BV421 (clone EH12.2H7) from BioLegend, and CD28-ECD (clone CD28-2) from Beckman Coulter. Flow cytometric acquisition and analysis of samples were performed on at least 100,000 events on an LSR II flow cytometer driven by the FACSDiva software package (BD Biosciences). Analyses of the acquired data were performed using FlowJo version 10.0.4 software (TreeStar).
Mavigner M., Zanoni M., Tharp G.K., Habib J., Mattingly C.R., Lichterfeld M., Nega M.T., Vanderford T.H., Bosinger S.E, & Chahroudi A. (2019). Pharmacological Modulation of the Wnt/β-Catenin Pathway Inhibits Proliferation and Promotes Differentiation of Long-Lived Memory CD4+ T Cells in Antiretroviral Therapy-Suppressed Simian Immunodeficiency Virus-Infected Macaques. Journal of Virology, 94(1), e01094-19.
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4

Multicolor Flow Cytometry of PBMCs

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Multicolor flow cytometric analysis was performed on whole blood or frozen PBMCs using predetermined optimal concentrations of the following fluorescently conjugated mAbs: CD3-PacBlue or -APC-Cy7 (clone SP34-2), CD95-PE-Cy5 (clone DX2), Ki-67-AF700 (clone B56), HLA-DR-PerCP-Cy5.5 (clone G46-6), CCR7-PE-Cy7 (clone 3D12), CCR5-PE or -APC (clone 3A9), CD45RA-FITC (clone L48), Biotin-CD122 (clone Mik-β3) from BD Biosciences; CD8-BV711 (clone RPA-T8), CD4-APC-Cy7 or -BV650 (clone OKT4), Streptavidin-PE from Biolegend, and CD28-ECD (clone CD28-2) from Beckman-Coulter. Flow cytometric acquisition and analysis of samples was performed on at least 100,000 events on an LSRII flow cytometer driven by the FACSDiva software package (BD Biosciences). Analyses of the acquired data were performed using FlowJo Version 10.0.4 software (TreeStar).
Mavigner M., Watkins B., Lawson B., Lee S.T., Chahroudi A., Kean L, & Silvestri G. (2014). Persistence of Virus Reservoirs in ART-Treated SHIV-Infected Rhesus Macaques after Autologous Hematopoietic Stem Cell Transplant. PLoS Pathogens, 10(9), e1004406.
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5

Multicolor Flow Cytometry Analysis

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After staining for surface antigens with mAbs at 4°C for 30 minutes, cells were incubated with 0.9% ammonium chloride for 5 minutes to lyse residual red blood cells. Cells were then extensively washed with PBS – 1% BSA and were run on a FACS Canto II® flow cytometer (BD Biosciences) with standard equipment. A minimum of 50,000 events was collected and acquired in list mode using the FACS Diva® software package (BD Biosciences).
Rutella S., Filippini P., Bertaina V., Li Pira G., Altomare L., Ceccarelli S., Brescia L.P., Lucarelli B., Girolami E., Conflitti G., Cefalo M.G., Bertaina A., Corsetti T., Moretta L, & Locatelli F. (2014). Mobilization of healthy donors with plerixafor affects the cellular composition of T-cell receptor (TCR)-αβ/CD19-depleted haploidentical stem cell grafts. Journal of Translational Medicine, 12, 240.
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6

Multicolor Flow Cytometry of Immune Subsets

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Multicolor flow cytometric analysis was performed on whole blood using predetermined optimal concentrations of the following fluorescently conjugated monoclonal antibodies: CD3-allophycocyanin (APC)-Cy7 (clone SP34–2), Ki-67-AF700 (clone B56), CCR7-fluorescein isothiocyanate (FITC) (clone A20), CD45RA-phycoerythrin (PE)-Cy7 (clone 5H9), and CD62L-PE (clone SK11) from BD Biosciences; CD8-Brilliant Violet 711 (BV711) (clone RPA-T8), CD4-BV650 (clone OKT4), and CD95-BV605 (clone DX2) from BioLegend; and CD28-PE-Cy5.5 (clone CD28.2) from Beckman Coulter. Flow cytometric acquisition and analysis of samples were performed on at least 100,000 events on an LSR II flow cytometer driven by the FACSDiva software package (BD Biosciences). Analyses of the acquired data were performed using FlowJo software (version 10.0.4; Tree Star).
Mavigner M., Liao L.E., Brooks A.D., Ke R., Mattingly C., Schoof N., McBrien J., Carnathan D., Liang S., Vanderford T.H., Paiardini M., Kulpa D., Lifson J.D., Dunham R.M., Easley K.A., Margolis D.M., Perelson A.S., Silvestri G, & Chahroudi A. (2021). CD8 Lymphocyte Depletion Enhances the Latency Reversal Activity of the SMAC Mimetic AZD5582 in ART-Suppressed Simian Immunodeficiency Virus-Infected Rhesus Macaques. Journal of Virology, 95(8), e01429-20.
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7

Multiparameter Flow Cytometry Analysis

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Frozen PBMCs were thawed using R10 medium (RPMI+L-glutamine+Penicillin Streptomycin+ 10% FCS) and subsequently stained with antibodies corresponding to either the chemokine/cytokine receptor, cytolytic, or transcription factor panels (see below). FoxP3 permeabilization kit (BD Pharmingen) was used for intracellular staining. Staining of the chemokine panel was carried out at 37°C. Samples were acquired on an LSRII flow cytometer (BD Biosciences) using the FACSDiva software package (BD Biosciences). Prior to each run, all samples were fixed in 2% PFA. Samples were then analyzed using the Flowjo software package (FlowJo, LLC). Gating strategies were developed based on florescence-minus-one (FMO) controls.
Phetsouphanh C., Aldridge D., Marchi E., Munier C.M., Meyerowitz J., Murray L., Van Vuuren C., Goedhals D., Fidler S., Kelleher A., Klenerman P, & Frater J. (2019). Maintenance of Functional CD57+ Cytolytic CD4+ T Cells in HIV+ Elite Controllers. Frontiers in Immunology, 10, 1844.
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8

Multicolor Flow Cytometric Immune Profiling

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Multicolor flow cytometric analysis was performed on whole blood and lymph node mononuclear cells using predetermined optimal concentrations of the following fluorescently conjugated monoclonal antibodies: CD3-APC-Cy7 (clone SP34–2), Ki-67-AF700 (clone B56), HLA-DR-PerCP-Cy5.5 (clone G46–6), CCR5-APC (clone 3A9, CCR7-FITC (clone A20 ), CD45RA-PE-Cy7 (clone 5H9), and CD62L-PE (clone SK11) from BD Biosciences; CD8-BV711 (clone RPA-T8), CD4-BV650 (clone OKT4), CD95-BV605 (clone DX2) and PD-1-BV421 (clone EH12.2H7) from Biolegend and CD28-PE-Cy5.5 (clone CD28.2) from Beckman Coulter. Flow cytometric acquisition and analysis of samples was performed on at least 100,000 events on an LSRII flow cytometer driven by the FACSDiva™ software package (BD Biosciences). Analyses of the acquired data were performed using FlowJo™ software (Tree Star, version 10.0.4).
Nixon C.C., Mavigner M., Sampey G.C., Brooks A.D., Spagnuolo R.A., Irlbeck D.M., Mattingly C., Ho P.T., Schoof N., Cammon C.G., Tharp G.K., Kanke M., Wang Z., Cleary R.A., Upadhyay A.A., De C., Wills S.R., Falcinelli S.D., Galardi C., Walum H., Schramm N.J., Deutsch J., Lifson J.D., Fennessey C.M., Keele B.F., Jean S., Maguire S., Liao B., Browne E.P., Ferris R.G., Brehm J.H., Favre D., Vanderford T.H., Bosinger S.E., Jones C.D., Routy J.P., Archin N.M., Margolis D.M., Wahl A., Dunham R.M., Silvestri G., Chahroudi A, & Garcia J.V. (2020). Systemic HIV and SIV latency reversal via non-canonical NF-κB signalling in vivo. Nature, 578(7793), 160-165.
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9

Dissecting SIV-Specific T-Cell Responses

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PBMCs from five ART-suppressed SIV-infected RMs were thawed. Pretreatment with AZD5582 was performed for 1 h at 100 nm. After two washes, PMA and ionomycin were added for 1 h at 500 ng/mL and 10 μg/mL, respectively. DMSO treatment controls were prepared in parallel. After 1 h, brefeldin-A (BFA) and Golgi stop solution were added following manufacturer’s recommendations (BD GolgiStop™ Protein Transport Inhibitor, BD Biosciences). Cells were incubated at 37°C, 5% CO2 in R10 for 6–8 h before staining with the following antibodies: CD3-APC-Cy7 (clone SP34–2), CD8-BV711 (clone RPA-T8), CD4-BV650 (clone OKT4), from Biolegend, IFN-γ-PE (clone B7), TNF-α-AF700 (clone Mab11) and IL-2-BV605 (clone MQ1–17H12) from BD Biosciences. Flow cytometric acquisition and analysis of samples was performed on at least 100,000 events on an LSRII flow cytometer driven by the FACSDiva software package (BD Biosciences). Analyses of the acquired data were performed using FlowJo™ (TreeStar, version 10.0.4) and Simplified Presentation of Incredibly Complex Evaluations (SPICE, version 6.0) software.
Nixon C.C., Mavigner M., Sampey G.C., Brooks A.D., Spagnuolo R.A., Irlbeck D.M., Mattingly C., Ho P.T., Schoof N., Cammon C.G., Tharp G.K., Kanke M., Wang Z., Cleary R.A., Upadhyay A.A., De C., Wills S.R., Falcinelli S.D., Galardi C., Walum H., Schramm N.J., Deutsch J., Lifson J.D., Fennessey C.M., Keele B.F., Jean S., Maguire S., Liao B., Browne E.P., Ferris R.G., Brehm J.H., Favre D., Vanderford T.H., Bosinger S.E., Jones C.D., Routy J.P., Archin N.M., Margolis D.M., Wahl A., Dunham R.M., Silvestri G., Chahroudi A, & Garcia J.V. (2020). Systemic HIV and SIV latency reversal via non-canonical NF-κB signalling in vivo. Nature, 578(7793), 160-165.
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10

Multicolor Flow Cytometric Immune Profiling

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Multicolor flow cytometric analysis was performed on whole blood and lymph node mononuclear cells using predetermined optimal concentrations of the following fluorescently conjugated monoclonal antibodies: CD3-APC-Cy7 (clone SP34–2), Ki-67-AF700 (clone B56), HLA-DR-PerCP-Cy5.5 (clone G46–6), CCR5-APC (clone 3A9, CCR7-FITC (clone A20 ), CD45RA-PE-Cy7 (clone 5H9), and CD62L-PE (clone SK11) from BD Biosciences; CD8-BV711 (clone RPA-T8), CD4-BV650 (clone OKT4), CD95-BV605 (clone DX2) and PD-1-BV421 (clone EH12.2H7) from Biolegend and CD28-PE-Cy5.5 (clone CD28.2) from Beckman Coulter. Flow cytometric acquisition and analysis of samples was performed on at least 100,000 events on an LSRII flow cytometer driven by the FACSDiva™ software package (BD Biosciences). Analyses of the acquired data were performed using FlowJo™ software (Tree Star, version 10.0.4).
Nixon C.C., Mavigner M., Sampey G.C., Brooks A.D., Spagnuolo R.A., Irlbeck D.M., Mattingly C., Ho P.T., Schoof N., Cammon C.G., Tharp G.K., Kanke M., Wang Z., Cleary R.A., Upadhyay A.A., De C., Wills S.R., Falcinelli S.D., Galardi C., Walum H., Schramm N.J., Deutsch J., Lifson J.D., Fennessey C.M., Keele B.F., Jean S., Maguire S., Liao B., Browne E.P., Ferris R.G., Brehm J.H., Favre D., Vanderford T.H., Bosinger S.E., Jones C.D., Routy J.P., Archin N.M., Margolis D.M., Wahl A., Dunham R.M., Silvestri G., Chahroudi A, & Garcia J.V. (2020). Systemic HIV and SIV latency reversal via non-canonical NF-κB signalling in vivo. Nature, 578(7793), 160-165.
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