Eclipse icyt flow cytometer, by Sony - Product details

1

FACS-Based Immune Cell Profiling

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All FACS assays were performed in 96-well round bottom plates (Corning). For neutralization assays, cells were harvested by trypsinization. For cell surface staining, cells were harvested by scraping at the indicated time points and fixed in 4% PFA (Thermo Fisher Scientific). Surface bound virus and 9C12 was detected using anti-human IgG-AF568 (Thermo Fisher, 1:1000). FcγRs were detected using anti-CD64-FITC (10.1, eBioscience, 0.2μg/sample), anti-CD32-PE (6C4, eBioscience, 25 ng/sample) and anti-CD16-APC, eBioCB16, eBioscience, 12 ng/sample). After antibody incubation, cells were washed three times with PBS-FBS and resuspended in 100 μL PBS-FCS.
Samples were acquired on a BD LSRII or a BD LSRFortessa flow cytometer (BD Biosciences).
For staining of whole blood, red blood cells were lysed using red blood cell lysis buffer (Miltenyi). After Fc-block (93, eBioscience, 1:100) white blood cells were stained with α-CD3 (17A2, BioLegend, 2.5 μg/mL), α-CD8a (KT15, BioRad, 0.125 μg/mL), H-2K^b-SIINFEKL tetramer (MBL International, 2 μL/sample) and viability dye (1:2500, eBioscience). Samples were acquired on an iCyt Eclipse flow cytometer (Sony Biotechnology).
All flow cytometry data was analyzed using FlowJo software (FlowJo, LLC).

Bottermann M., Foss S., Caddy S.L., Clift D., van Tienen L.M., Vaysburd M., Cruickshank J., O’Connell K., Clark J., Mayes K., Higginson K., Lode H.E., McAdam M.B., Sandlie I., Andersen J.T, & James L.C. (2019). Complement C4 Prevents Viral Infection through Capsid Inactivation. Cell Host & Microbe, 25(4), 617-629.e7.

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2

Single-Cell RNA-Seq Library Preparation

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For analysis by 10X Genomics, tubes were defrosted and gently mixed, and 1.7 ml of the samples were transferred into an Eppendorf Lowbind tube and centrifuged at 4°C for 3 min at 3000g. The PBS/methanol mix was discarded and replaced by 400 µl of PBS. Cell concentration was measured using an iCyt Eclipse flow cytometer (SONY), based on forward scatter. Cell concentration ranged from 1044 cells ml⁻¹ to 9855 cells ml⁻¹. All concentrations were brought to 1000 cells ml⁻¹ to target 7000 cells recovery, according to the 10X Genomics Cell Suspension Volume Calculator Table provided in the user guide. Two libraries were prepared and sequenced on different occasions: B4T19 and B7T17 in January 2020 and B3T15, B3T20, B4T13, B4T15, B4T20, B6T17, B7T16, and B7T18 in August 2020. All the following steps for library preparation were accomplished according to the manufacturer’s protocol for Chromium Next GEM Single Cell 3’ Reagent Kit v3.1. Libraries were sequenced using NextSeq^® 500 High Output kit (75 cycles).
Raw 10X reads were trimmed using TrimGalore (v. 0.6.5), a Cutadapt wrapper³⁹, and the poly-A tail was removed.

Fromm A., Hevroni G., Vincent F., Schatz D., Martinez-Gutierrez C.A., Aylward F.O, & Vardi A. (2023). Homing in on the rare virosphere reveals the native host of giant viruses. bioRxiv.

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3

Flow Cytometry Cell Acquisition

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Sample acquisition was performed using an iCyt Eclipse Flow Cytometer (Sony Biotechnology, Inc.) with a minimum of 2.0 × 10⁴ cells acquired per sample tube. The data were analyzed using iCyt EC800 software, version 1.3.5.

Afshar M., Richards S., Mann D., Cross A., Smith G.B., Netzer G., Kovacs E, & Hasday J. (2014). Acute Immunomodulatory Effects of Binge Alcohol Ingestion. Alcohol (Fayetteville, N.Y.), 49(1), 57-64.

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4

Quantifying Phytoplankton and Bacteria by Flow Cytometry

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Flow cytometry analyses were performed on Eclipse iCyt flow cytometer (Sony Biotechnology Inc, Champaign, IL, USA) equipped with 405 and 488 nm solid-state air-cooled lasers, and with standard optic filter set-up. E. huxleyi cells were identified by plotting the chlorophyll fluorescence (663–737 nm) against side scatter and were quantified by counting the high-chlorophyll events. For bacterial counts, samples were fixed with a final concentration of 0.5% glutaraldehyde for at least 30 min at 4°C, then plunged into liquid nitrogen and stored at −80°C until analysis. After thawing, samples were stained with SYBR gold (Invitrogen) that was diluted 1:10,000 in Tris–EDTA buffer, incubated for 20 min at 80°C, and cooled to room temperature. Samples were analyzed by flow cytometry (ex: 488 nm; em: 500–550 nm).

Barak-Gavish N., Dassa B., Kuhlisch C., Nussbaum I., Brandis A., Rosenberg G., Avraham R, & Vardi A. (2023). Bacterial lifestyle switch in response to algal metabolites. eLife, 12, e84400.

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5

Flow Cytometry Analysis of Phytoplankton

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Flow cytometry analyses were performed using an Eclipse iCyt flow cytometer (Sony Biotechnology Inc., Champaign, IL, USA) equipped with 405- and 488-nm solid-state air-cooled lasers and with a standard optic filter setup. E. huxleyi cells were identified by plotting the chlorophyll fluorescence (663 to 737 nm) against side scatter and were quantified by counting the high-chlorophyll events. For bacterial counts, samples were fixed with a final concentration of 0.5% glutaraldehyde for at least 30 min at 4°C, then plunged into liquid nitrogen, and stored at −80°C until analysis. After thawing, samples were stained with SYBR Gold (Invitrogen) that was diluted 1:10,000 in tris-EDTA buffer, incubated for 20 min at 80°C, and cooled to room temperature. Samples were analyzed by flow cytometry (excitation, 488 nm; emission, 500 to 550 nm). For algal cell death analysis, samples were stained with a final concentration of 1 μM SYTOX Green (Invitrogen), incubated in the dark for 30 min at room temperature, and analyzed by flow cytometry (excitation, 488 nm; emission, 500 to 550 nm). An unstained sample was used as control to eliminate the background signal.

Barak-Gavish N., Frada M.J., Ku C., Lee P.A., DiTullio G.R., Malitsky S., Aharoni A., Green S.J., Rotkopf R., Kartvelishvily E., Sheyn U., Schatz D, & Vardi A. (2018). Bacterial virulence against an oceanic bloom-forming phytoplankter is mediated by algal DMSP. Science Advances, 4(10), eaau5716.

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6

Quantifying Algae and Viruses in Marine Samples

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Cells were quantified using a Multisizer 4 Coulter Counter (Beckman Coulter, version 4.01) and an Eclipse (iCyt) flow cytometer (Sony Biotechnology, Champaign, IL, USA, using ec800 version 1.3.7 software), equipped with 405 and 488 nm solid state-air cooled lasers (both 25 mW on the flowCell) and standard filter setup. Algae were identified by plotting chlorophyll autofluorescence in the red channel (737 to 663 nm) versus green fluorescence (500 to 550 nm) or side scatter (Supplementary Fig. 15a). Extracellular viral particles were quantified as described previously (Supplementary Fig. 15b)24 (link).
Quantification of E. huxleyi and bacterial cells from agarose samples was performed by suspending agarose samples in ASW. A sterile biopsy punch (8 mm diameter) was used for sampling. Each punched sample was added to 500 µL ASW, vortexed, pipetted and placed at 18 °C for 15 min to allow transfer of cells from the agarose to the ASW. Samples were then filtered through a 35 µm cell strainer. Plaques were smaller than 8 mm in diameter, and hence contained also uninfected areas. E. huxleyi cells were quantified as described above. Bacterial cells were quantified as described previously (Supplementary Fig. 15b)54 .

Schleyer G., Shahaf N., Ziv C., Dong Y., Meoded R.A., Helfrich E.J., Schatz D., Rosenwasser S., Rogachev I., Aharoni A., Piel J, & Vardi A. (2019). In plaque-mass spectrometry imaging of a bloom-forming alga during viral infection reveals a metabolic shift towards odd-chain fatty acid lipids. Nature microbiology, 4(3), 527-538.

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7

Quantifying Algae and Viruses in Marine Samples

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Cells were quantified using a Multisizer 4 Coulter Counter (Beckman Coulter, version 4.01) and an Eclipse (iCyt) flow cytometer (Sony Biotechnology, Champaign, IL, USA, using ec800 version 1.3.7 software), equipped with 405 and 488 nm solid state-air cooled lasers (both 25 mW on the flowCell) and standard filter setup. Algae were identified by plotting chlorophyll autofluorescence in the red channel (737 to 663 nm) versus green fluorescence (500 to 550 nm) or side scatter (Supplementary Fig. 15a). Extracellular viral particles were quantified as described previously (Supplementary Fig. 15b)24 (link).
Quantification of E. huxleyi and bacterial cells from agarose samples was performed by suspending agarose samples in ASW. A sterile biopsy punch (8 mm diameter) was used for sampling. Each punched sample was added to 500 µL ASW, vortexed, pipetted and placed at 18 °C for 15 min to allow transfer of cells from the agarose to the ASW. Samples were then filtered through a 35 µm cell strainer. Plaques were smaller than 8 mm in diameter, and hence contained also uninfected areas. E. huxleyi cells were quantified as described above. Bacterial cells were quantified as described previously (Supplementary Fig. 15b)54 .

Schleyer G., Shahaf N., Ziv C., Dong Y., Meoded R.A., Helfrich E.J., Schatz D., Rosenwasser S., Rogachev I., Aharoni A., Piel J, & Vardi A. (2019). In plaque-mass spectrometry imaging of a bloom-forming alga during viral infection reveals a metabolic shift towards odd-chain fatty acid lipids. Nature microbiology, 4(3), 527-538.

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8

Flow cytometry for cell abundance

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Flow cytometry measurements (cell abundance and Sytox staining) were obtained using Eclipse iCyt flow cytometer (Sony Biotechnology Inc., Champaign, IL, United States), equipped with a 488nm solid state air cooled 25mW laser with a standard filter setup. Cells were identified by plotting chlorophyll fluorescence in the red channel (737–663nm) vs. green fluorescence (500–550nm) or forward scatter. At least 5,000 cells were analyzed per sample, with at least three biological replicates.

Graff van Creveld S., Ben-Dor S., Mizrachi A., Alcolombri U., Hopes A., Mock T., Rosenwasser S, & Vardi A. (2021). Biochemical Characterization of a Novel Redox-Regulated Metacaspase in a Marine Diatom. Frontiers in Microbiology, 12, 688199.

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Eclipse icyt flow cytometer

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8 protocols using eclipse icyt flow cytometer

FACS-Based Immune Cell Profiling

Single-Cell RNA-Seq Library Preparation

Flow Cytometry Cell Acquisition

Quantifying Phytoplankton and Bacteria by Flow Cytometry

Flow Cytometry Analysis of Phytoplankton

Quantifying Algae and Viruses in Marine Samples

Quantifying Algae and Viruses in Marine Samples

Flow cytometry for cell abundance

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