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Icyt synergy sorter system

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The ICyt Synergy sorter system is a high-performance flow cytometry instrument designed for cell sorting applications. It features advanced optics and electronics to enable efficient and accurate cell separation. The core function of the ICyt Synergy sorter system is to facilitate the isolation and purification of specific cell populations from complex biological samples.

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Lab products found in correlation

Winlist 3d 8, by Verity Software House (1 mentions) A1rsi confocal microscope, by Nikon (1 mentions) Proteinase k, by Qiagen (1 mentions) Lsrii flow cytometer, by BD (1 mentions) Rpmi 1640 medium, by Corning (1 mentions)

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ICyt Synergy

4 protocols using icyt synergy sorter system

1

Quantifying CD3+ Cells in Healing Ear Tissue

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To quantify the number of CD3+ cells present in healing ear tissue, tissue was harvested from a separate group of Mm-UKY and Ac females at D0, 1, 3, 7, and 15 using an 8 mm biopsy punch. Harvested tissue from both ears was combined and a single-cell suspension was created using a combination of enzymatic and mechanical digestion, as previously described (16 (link)). Total cells were counted by hemacytometer and incubated with PE-conjugated-anti-CD3 (Clone 17A2, BioLegend, San Diego, CA) at a concentration of 1 μg/106 cells for 1 hour at room temperature, washed and suspended in cell staining buffer (Cat#420201, BioLegend). Flow cytometry was carried out at the University of Kentucky Flow Cytometry Core using the iCyt Synergy sorter system (Sony Biotechnology Inc., San Jose, CA). Laser calibration and compensation was performed for each experiment using unstained and single fluorescent control samples. Analysis was done using FlowJo (Version 10, FlowJo, LLC, Ashland, OR) to identify CD3-positive lymphocytes by PE fluorescence and forward- and side-scatter. The same gating strategies between species were used (n = 4 or 5 animals per timepoint).
Gawriluk T.R., Simkin J., Hacker C.K., Kimani J.M., Kiama S.G., Ezenwa V.O, & Seifert A.W. (2020). Complex Tissue Regeneration in Mammals Is Associated With Reduced Inflammatory Cytokines and an Influx of T Cells. Frontiers in Immunology, 11, 1695.
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2

Mitochondrial Membrane Potential Analysis

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MDS-L cells (7.5 × 105 cells/ml) were exposed to WFA (10 μM) or DMSO for 8 h. Cells treated with FCCP (50 μM) for 2 h were used as a positive control. JC-1 was added at a 1 μM final concentration to cells for the last 30 min of treatment at 37°C and fluorescence was measured by the iCyt Synergy sorter system (Sony Biotechnology Inc., San Jose, CA) with 488 and 561 nm lasers. WinList 3d 8.0 software (Verity Software House Inc., Topsham, Maine) was used for data analyses. For microscopy, cells treated with WFA (10 μM) or DMSO and stained with JC-1 as described above were mounted on poly-l Lysine (MilliporeSigma-Aldrich # P-6282) coated slides by Cytospin. Pictures were taken on the same day with a Nikon A1RSi confocal microscope (Nikon Instruments Inc, Melville, NY).
Oben K.Z., Alhakeem S.S., McKenna M.K., Brandon J.A., Mani R., Noothi S.K., Jinpeng L., Akunuru S., Dhar S.K., Singh I.P., Liang Y., Wang C., Abdel-Latif A., Stills Jr H.F., St. Clair D.K., Geiger H., Muthusamy N., Tohyama K., Gupta R.C, & Bondada S. (2017). Oxidative stress-induced JNK/AP-1 signaling is a major pathway involved in selective apoptosis of myelodysplastic syndrome cells by Withaferin-A. Oncotarget, 8(44), 77436-77452.
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3

Quantifying Murine T-Cell Receptor Excision Circles

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CD4+ and CD8+ splenocytes were sorted with an iCyt Synergy sorter system (Sony). Cell lysates were prepared by incubation with proteinase K (QIAGEN) at 55˚C for one hour, followed by inactivation at 95˚C for 15 minutes. The lysate from 50,000 cells/5μL was added to a real-time quantitative polymerase chain reaction (PCR). The primers and reaction program were described formerly.49 (link) The standard curves for murine TRECs were generated by using a standard mouse TRECs plasmid, which was a generous gift from Dr. Gregory D. Sempowski, Duke University Medical Center.49 (link) For each real-time PCR assay, stock dilutions of 107, 106, 105, 104, 103 and 102 plasmids per 5uL were run in triplicate to generate standard curves. TREC frequency of isolated cells was determined in triplicate.
Zheng Z., Ai J., Guo L., Ye X., Bondada S., Howatt D., Daugherty A, & Li X.A. (2018). Scavenger receptor BI is critical in maintaining normal T cell development and enhancing thymic regeneration. Arteriosclerosis, thrombosis, and vascular biology, 38(11), 2706-2717.
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4

Isolation and Characterization of Murine Lymphocyte Subsets

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Single-cell lymphocyte suspensions from mouse tissues were prepared by pressing tissues through a 40µm strainer using the plunger end of a syringe in Hank’s buffered salt solution (HBSS) (Millipore Sigma-Aldrich). Tibiae and femora were flushed with a 26G syringe in HBSS. Peritoneal cells were obtained by peritoneal lavage with HBSS. Cells were resuspended in RPMI 1640 medium (Corning, New York, NY) supplemented with 10% FBS. For multi-color immunofluorescence analysis, single-cell suspensions of mononuclear cells were first incubated with normal rat IgG (10µg/106 cells) at 4°C for 15 min to block Fcγ receptors. The cells were then labeled with fluorochrome conjugated anti-mouse antibodies for 30 minutes on ice. Stained cells were analyzed using Becton Dickinson LSRII flow cytometer and CellQuest Pro software. Anti-CD19, anti-CD11b and anti-CD5 were used to identify and sort B-1a (CD19+CD5+CD11b+), B-1b (CD19+CD5-CD11b+), B-2 (CD19+CD5-CD11b-) cells from the peritoneum of C57BL/6J mice using iCyt Synergy sorter system from (Sony Biotechnology, San Jose, CA). Anti-CD19 and anti-CD90.2 antibodies were used to identify and sort T-cells (CD19-CD90.2+). Intracellular staining of IL-10 and IFN-γ was performed according to Biolegend protocol after a short-term stimulation with PMA and Ionomycin for 4 hours.
Alhakeem S.S., McKenna M.K., Oben K.Z., Noothi S.K., Rivas J.R., Hildebrandt G.C., Fleischman R.A., Rangnekar V.M., Muthusamy N, & Bondada S. (2018). Chronic lymphocytic leukemia derived interleukin-10 suppresses anti-tumor immunity. Journal of immunology (Baltimore, Md. : 1950), 200(12), 4180-4189.
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