Sp6800 spectral cell analyzer

Manufactured by Sony

Sourced in United States

The SP6800 Spectral Cell Analyzer is a laboratory instrument designed for the analysis and characterization of cell samples. It utilizes advanced spectral flow cytometry technology to provide detailed information about the physical and fluorescent properties of individual cells within a sample. The core function of the SP6800 is to accurately measure and analyze multiple parameters of cells, including size, granularity, and the presence of specific fluorescent markers.

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16 protocols using sp6800 spectral cell analyzer

Quantification of Antigen-Specific CD8+ T Cells

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Allophycocyanin-labelled dextramers specific for OVA H-2Kb (257-SIINFEKL-264) were purchased from Immudex (Copenhagen, Denmark). Samples for flow cytometry were stained with the appropriate antibody cocktails in ice-cold PBS supplemented with 2 mM EDTA and 1% FBS. Anti-mouse CD45 (clone 30F11), CD8α (clone 53-6.7), CD103 (clone 2E7), CD44 (clone IM7), CD45.1 (clone A20) and CD45.2 (clone 104) antibodies were obtained from eBioscience. Anti-mouse CD62L (clone MEL-14) and CD69 (clone H1.2F3) antibodies were obtained from BD Biosciences. Anti-mouse CD279 (PD-1, clone 29F.1A12) was obtained from Biolegend. Events were acquired using an LSRFortessa SORP (Becton Dickinson) flow cytometer or Spectral Cell Analyzer SP6800 (Sony) and data were analysed using FlowJo V10 software (Tree Star).

Enamorado M., Iborra S., Priego E., Cueto F.J., Quintana J.A., Martínez-Cano S., Mejías-Pérez E., Esteban M., Melero I., Hidalgo A, & Sancho D. (2017). Enhanced anti-tumour immunity requires the interplay between resident and circulating memory CD8+ T cells. Nature Communications, 8, 16073.

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Single-Cell CD3+ Cell Isolation

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Single-Cell suspensions were cleared of dead cells using the Dead-Cell Removal Kit (Miltenyi Biotech, Bergisch-Gladbach, Germany) and enriched for CD3⁺ cells by CD3-MACS enrichment (Miltenyi Biotech, Bergisch-Gladbach, Germany). Isolated cells were then incubated with VivaFix™ 398/550 (BioRad Laboratories, CA, USA) according to the manufacturer´s instructions, followed by fixation in 4% paraformaldehyde (PFA) for 10 min. After centrifugation (350×g; 4 °C; 5 min), the cell pellet was suspended in 0.5 ml 4 °C FACS buffer. Cell suspensions were then centrifuged at 350×g for 5 min, followed by resuspension in FACS buffer, twice. Finally, cells were resuspended in 0.5–1 ml of FACS buffer, depending on cell count. Sorting was carried out using a Spectral cell Analyzer (SP6800, Sony Biotechnology, CA, USA) in standardization mode, with photomultiplier tube voltage set to maximum in order to reach a saturation rate below 0.1%. Gating was performed using FCS Express 7 plus (De Novo Software, CA, USA) at the Lighthouse Core Facility, University of Freiburg.

Ravi V.M., Neidert N., Will P., Joseph K., Maier J.P., Kückelhaus J., Vollmer L., Goeldner J.M., Behringer S.P., Scherer F., Boerries M., Follo M., Weiss T., Delev D., Kernbach J., Franco P., Schallner N., Dierks C., Carro M.S., Hofmann U.G., Fung C., Sankowski R., Prinz M., Beck J., Salié H., Bengsch B., Schnell O, & Heiland D.H. (2022). T-cell dysfunction in the glioblastoma microenvironment is mediated by myeloid cells releasing interleukin-10. Nature Communications, 13, 925.

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Multiparametric Flow Cytometry of PBMCs

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PBMCs were stained in four different panels: T-cell maturation, activation and regulation, and B-cell maturation/activation (presented elsewhere). Definition of the maturation stages used for both T and B cells has been published previously [24, 25] . The staining process is described in the supplementary section.
Samples were analyzed on a Sony Spectral Cell Analyzer SP6800 (Sony Corporation, Tokyo, Japan) flow cytometer. The software SP6800 version 1.6.3.7151 was used for acquisition and spectral unmixing (weighted least-squares algorithm). Cell population gating was performed on unmixed data in FlowJo v.10.4 (FlowJo LLC, Ashland, USA) as presented elsewhere.
Copyright © 2019 Wolters Kluwer Health, Inc. Unauthorized reproduction of this article is prohibited.

Hønge B.L., Petersen M.S., Jespersen S., Medina C., Té D.D.S., Kjerulff B., Engell-Sørensen T., Madsen T., Laursen A.L., Wejse C., Krarup H., Møller B.K, & Erikstrup C. (2019). T-cell and B-cell perturbations identify distinct differences in HIV-2 compared with HIV-1-induced immunodeficiency. AIDS (London, England), 33(7).

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Multiparametric Flow Cytometry of Immune Cells

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Single-cell suspensions of skin and dLN cells were incubated with anti-CD16/CD32 (TONBO Biosciences) for 5 min, and then stained using the fluorochrome-conjugated antibodies described in Table S1. Intracellular GzmB and CTLA-4 were stained using Fixation Buffer and 10 × Permeabilization Wash Buffer (BioLegend) as described in the manufacturer's protocol. The fluorescence intensity of KikGR-Red was reduced by fixation, thus, the proportion of KikGR-Red⁺ cells was lower in samples subjected to intracellular staining than in fresh samples. Dead cells were stained with 7-AAD (eBioscience) or propidium iodide (Wako) and excluded from our analyses. Stained samples were analyzed using a SP6800 Spectral Cell Analyzer (SONY). All data were exported as FCS files and analyzed using FlowJo software (Tree Star).

Ikebuchi R., Fujimoto M., Nakanishi Y., Okuyama H., Moriya T., Kusumoto Y, & Tomura M. (2019). Functional Phenotypic Diversity of Regulatory T Cells Remaining in Inflamed Skin. Frontiers in Immunology, 10, 1098.

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Cell Cycle and Apoptosis Analysis

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Cell-cycle analysis was performed by propidium iodide (PI; Sigma-Aldrich 537,060) staining to quantify the sub-G1, S and G2 population, knowing that it can reflect the extent of cell cycle. Briefly, 1 × 10⁵ cells were seeded in a 6-well plate. After 24 h, cells were treated with the indicated agents for 72 h, fixed with 70% ethanol and stained with PI (50 µg/mL). The stained cells were tested by SP6800 Spectral Cell Analyzer (Sony) and analyzed by using the FlowJo software. For apoptosis assay, cells were treated with the indicated compounds and stained with 5ul FITC-Annexin V (BD Biosciences 556,419) and 5ul PI (BD Biosciences 556,463). After 15 min, the stained cells were analyzed by flow cytometry.

Han Z., Jia Q., Zhang J., Chen M., Wang L., Tong K., He W., Zhang Y., Zhu W., Qin J., Wang T., Liu T., Ma Y., Chen Y., Zha S, & Zhang C. (2023). Deubiquitylase YOD1 regulates CDK1 stability and drives triple-negative breast cancer tumorigenesis. Journal of Experimental & Clinical Cancer Research : CR, 42, 228.

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Multiparametric Flow Cytometry Analysis of Cryopreserved PBMCs

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Frozen PBMCs were thawed in a warm bath and then placed in 10 mL of a solution of RPMI complete (RPMI 1640 medium (Gibco) supplemented with 10% fetal bovine serum, 2 mmol/l l-glutamine, 1% penicillin/streptomycin, 1% minimal essential media nonessential amino acids, 1 mmol/l sodium pyruvate, and 0.1 mmol/l 2-mercaptoethanol) with DNase I (Sigma-Aldrich). PBMCs were washed once in phosphate-buffered saline (PBS) with ethylenediaminetetraacetic acid (EDTA) 2 mM and stained with Zombie NIR fixable viability kit (BioLegend) prior to staining for flow cytometry. Stained cells were fixed with 1% formalin (Sigma) and analyzed with an SP6800 Spectral Cell Analyzer (SONY).
The following antibodies were used for human cells. From BioLegend: aCD3-PE clone OKT3, aCD19-PE clone HIB19, aCD33-PE-Cy7 clone WM53, aCD15-PB clone MMA, aCD14-FITC clone M5E2, aCD16-BV785 clone 3G8, aHLA-DR-BV570 clone L243, aPD1-PE clone EH12.2H7, aCD137-PE-Cy7 clone 4B4-1, aCD8-PB clone HIT8a, CX3CR1-BV421 clone 2A9-1, aCCR3-PE clone 5E8, aCD14-BV605 clone M5E2, aCD4-PB clone OKT4, aCD25-BV421 clone BC96, CCR2-BV510 clone K036C2, CXCR2-PerCP-Cy5.5 clone 5E8, aCD33-FITC clone P67.6. From BD Biosciences: aCD3-PE-CF594 clone UCHT1, aCD45-PE-CF594 clone HI30, aCD4-BV510 clone SK3.

Cardot-Ruffino V., Bollenrucher N., Delius L., Wang S.J., Brais L.K., Remland J., Keheler C.E., Sullivan K.M., Abrams T.A., Biller L.H., Enzinger P.C., McCleary N.J., Patel A.K., Rubinson D.A., Schlechter B., Slater S., Yurgelun M.B., Cleary J.M., Perez K., Dougan M., Ng K., Wolpin B.M., Singh H, & Dougan S.K. (2023). G-CSF rescue of FOLFIRINOX-induced neutropenia leads to systemic immune suppression in mice and humans. Journal for Immunotherapy of Cancer, 11(6), e006589.

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Multiparameter Flow Cytometry Panel

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Cells were stained for 20 min at 4°C in PBS + 5% BSA with the following antibodies from eBioscience at 1:300 dilutions: Anti-CD8-PerCP5.5 (45–0081-82), anti-CD45.1-PE (12–0453-82), anti-CD45.2-FITC (11–0454-82), anti-TCR-Va2-APC (17–5812-82), anti-CD44-PE-Cy7 (25–0441-82), and anti-CD62L-BV605 (104437, BioLegend). Samples were acquired on a SP6800 Spectral Cell Analyzer (Sony Biotechnology) and analyzed using FlowJo 10.1r7 software (FlowJo, LLC).

Liedmann S., Liu X., Guy C.S., Crawford J.C., Rodriguez D.A., Kuzuoğlu-Öztürk D., Guo A., Verbist K.C., Temirov J., Chen M.J., Ruggero D., Zhang H., Thomas P.G, & Green D.R. (2022). Localization of a TORC1-eIF4F translation complex during CD8+ T cell activation drives divergent cell fate. Molecular cell, 82(13), 2401-2414.e9.

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NAD(P)H Sensor Protocol for Flow Cytometry

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After surface staining, 500,000 cells were incubated for 30 min at 37 °C in PBS supplemented with a JZL1707 NAD(P)H sensor according to the manufacturer’s instructions (AAT Bioquest, Euromedex, Souffelweyersheim, France). Cells were subsequently washed with PBS and the fluorescence intensity of the JZL1707 NAD(P)H sensor was read in the PE channel on a SP6800 Spectral Cell Analyzer (Sony, Weybridge, UK) and analyzed with FlowJo V10 software (BD Biosciences, Le Pont de Claix, France).

Khou S., Popa A., Luci C., Bihl F., Meghraoui-Kheddar A., Bourdely P., Salavagione E., Cosson E., Rubod A., Cazareth J., Barbry P., Mari B., Rezzonico R., Anjuère F, & Braud V.M. (2020). Tumor-Associated Neutrophils Dampen Adaptive Immunity and Promote Cutaneous Squamous Cell Carcinoma Development. Cancers, 12(7), 1860.

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Quantifying Cell Death by PI and Annexin V

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PI staining was done to quantify the sub-G1 population, which can reflect the extent of cell death. Cells were seeded in 6-well plates 24 h prior to treatment with different compounds. After 72 h, cells then collected, fixed and stained with PI (50 μg/mL). The stained cells were analyzed by SP6800 Spectral Cell Analyzer (Sony) and quantified by using the FlowJo software.
Cell death was also detected by flow cytometry using Annexin V-FITC/PI double staining (BD Biosciences) using the manufacturer’s protocol. The stained cells were immediately analyzed by flow cytometric analysis.

Liu L., Deng P., Liu S., Hong J.H., Xiao R., Guan P., Wang Y., Wang P., Gao J., Chen J., Sun Y., Chen J., Mai H.Q, & Tan J. (2023). Enhancer remodeling activates NOTCH3 signaling to confer chemoresistance in advanced nasopharyngeal carcinoma. Cell Death & Disease, 14(8), 513.

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SIgA Conjugation and Bacterial Binding

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One hundred μg of SIgA and derived fractions were conjugated with DyLight 488 and characterized according to manufacturer’s instructions (Thermo Fisher Scientific). About 5 μg of individual DyLight 488-labeled SIgA preparations, previously confirmed as oversaturating the bacteria, were incubated with 10⁶ CFU of E. coli O55 for 30 min, followed by washing with 1% FBS in PBS and analyzing by flow cytometry using SP6800 Spectral Cell Analyzer (Sony Biotechnology, San Jose, CA).

Raskova Kafkova L., Brokesova D., Krupka M., Stehlikova Z., Dvorak J., Coufal S., Fajstova A., Srutkova D., Stepanova K., Hermanova P., Stepankova R., Uberall I., Skarda J., Novak Z., Vannucci L., Tlaskalova-Hogenova H., Jiraskova Zakostelska Z., Sinkora M., Mestecky J, & Raska M. (2020). Secretory IgA N-glycans contribute to the protection against E. coli O55 infection of germ-free piglets. Mucosal Immunology, 14(2), 511-522.

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