Facsymphony s6 cell sorter

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The BD FACSymphony S6 cell sorter is a high-performance flow cytometry instrument designed for advanced cell analysis and sorting. It features a compact design and offers multiparameter detection capabilities.

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9 protocols using facsymphony s6 cell sorter

Multiparametric Cell Sorting of Cerebrovascular Cells

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Tissue was cut and minced on ice, and then enzymatically digested twice with 1 mg/ml Collagenase Type IV (MilliporeSigma, Darmstadt, Germany) and 100 μg/ml DNase I (MilliporeSigma) at 37℃ for 20 min and 30 min, respectively. The cell suspension was filtered, washed, and pelleted. Cells were then resuspended with 25% Percoll and underwent 20 min centrifugation without break. The top layer containing cell debris and myelin was removed. A multispectral LED light was used to perform a 30-min irradiation treatment to reduce background autofluorescence [23 ]. Cells were stained with an anti-human CD31-PE (303,105, BioLegend, San Diego, United States), CD45-BV421 (304,031, BioLegend), CD13-PE-Cy7 (301,711, BioLegend), P2RY12-FITC (392,107, BioLegend), CD49f-PerCP-Cy5.5 (313,617, BioLegend), CD90-BV711 (328,139, BioLegend) and GLAST-APC (130–123-555, Miltenyi Biotec, Bergisch Gladbach, Germany) antibody cocktail. Size, granularity, and antibody-specific gating were set to sort ECs, pericytes, microglia and neuroglia using the FACSymphony S6 Cell Sorter (BD Biosciences, Franklin Lakes, United States) (Fig. S1a).

Li Y., Girard R., Srinath A., Cruz D.V., Ciszewski C., Chen C., Lightle R., Romanos S., Sone J.Y., Moore T., DeBiasse D., Stadnik A., Lee J.J., Shenkar R., Koskimäki J., Lopez-Ramirez M.A., Marchuk D.A., Ginsberg M.H., Kahn M.L., Shi C, & Awad I.A. (2024). Transcriptomic signatures of individual cell types in cerebral cavernous malformation. Cell Communication and Signaling : CCS, 22, 23.

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Purification and Sorting of CD8+ T Lymphocytes

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CD8⁺ T lymphocytes were purified from human buffy coats as described in Sections 2.1 and 2.2 and the cells were sorted according to their CD8 expression (anti‐CD8‐V450, clone: RPA‐T8, BD Biosciences, Heidelberg, Germany, RRID: AB_1645581) into CD8^Low and CD8^High T cell subsets with the FACSymphony S6 Cell sorter (BD Biosciences, San Diego, CA). A general gating strategy for the CD8⁺ T lymphocytes (Supplementary Figure S2A) is provided in the supporting information. As described elsewhere¹⁴, ¹⁵ the sorting samples were compared to a donor‐matching 10 % autologous serum control (Supplementary Figure S2B and C).

Burkard T., Herrero San Juan M., Dreis C., Kiprina A., Namgaladze D., Siebenbrodt K., Luger S., Foerch C., Pfeilschifter J.M., Weigert A, & Radeke H.H. (2022). Differential expression of CD8 defines phenotypically distinct cytotoxic T cells in cancer and multiple sclerosis. Clinical and Translational Medicine, 12(12), e1068.

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Expansion and Sorting of NK Cell Subsets

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CD56⁺ cells prepared from human PBMCs were cultured with mitomycin C-treated K562 cell line in the presence of anti-NKG2C antibody (10 µg/mL, clone: 134522, R&D systems), IL-2 (100 IU/mL, Peprotech) and IL-15 (10 ng/mL, Peprotech) for one week^{53 (link)}. Then, LAG-3⁻PD-1⁻, LAG-3⁺PD-1⁻ and LAG-3⁺PD-1⁺ NK cells were sorted with a FACSymphony™ S6 Cell Sorter (BD Biosciences) and co-cultured with CD4⁺ T cells in the presence of mogamulizumab.

Maeda Y., Wada H., Sugiyama D., Saito T., Irie T., Itahashi K., Minoura K., Suzuki S., Kojima T., Kakimi K., Nakajima J., Funakoshi T., Iida S., Oka M., Shimamura T., Doi T., Doki Y., Nakayama E., Ueda R, & Nishikawa H. (2021). Depletion of central memory CD8+ T cells might impede the antitumor therapeutic effect of Mogamulizumab. Nature Communications, 12, 7280.

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Isolation and Characterization of Peripheral Blood Lymphocytes

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Cells were isolated from fresh peripheral blood by density gradient centrifugation using lymphocyte separation media (MP Biomedicals, #0850494-CF), washed in PBS, and counted. Approximately 5e6 cells were then resuspended in 100μl PBS. Cells were stained with LIVE/DEAD amine-reactive viability dye (Thermo Fisher Scientific, #L34966) and a panel of antibodies, including anti-CD3 alexa fluor 700 (clone SP43–2, BD Biosciences, #557917), anti-CD4 allophycocyanin (clone L200, BD, #551980), anti-CD8α pacific blue (clone RPA-T8, BD, #558207), CD28 energy-coupled dye [ECD] (clone 28.2, Beckman Coulter, Brea, CA, #6607111), CD95 Cy5-PE (clone DX2, BD, #561977), for 20 minutes at 4°C. Cells were washed in PBS and resuspended in complete RPMI with 10% FBS. For each population, 30,000 cells were sorted using a FACSymphony S6 cell sorter (BD).

Rahmberg A.R., Markowitz T.E., Mudd J.C., Hirsch V, & Brenchley J.M. (2022). Epigenetic reprogramming leads to down regulation of CD4 and functional changes in African green monkey memory CD4+ T cells. Journal of immunology (Baltimore, Md. : 1950), 209(2), 337-345.

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Single-Cell Isolation and Clonal Expansion

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Seventy-two hours after transfection, cells were detached by trypsin (25200056; Life Technologies) and counted with a countess automated cell counter (Invitrogen). Ten thousand cells were resuspended in PBS (1X; pH 7.4; 10010-023; Thermo Fisher Scientific, Inc) containing 20% fetal bovine serum (Atlanta Biologicals) and treated with DNA-staining DAPI (D3571; Thermo Fisher Scientific, Inc) prior to sorting by FACS with a BD FACSymphony S6 Cell Sorter (BD Sciences). Forward scatter (FSC)-area and side scatter (SSC)-area settings were used to gate for intact cells and exclude debris. For singlet isolation, the samples were gated by SSC-height and SSC-width. To further discriminate against doublet events, the cells were subsequently gated using FSC-height and FSC-width parameters. Following debris and doublet exclusion, live singlets were gated using 4′,6-diamidino-2-phenylindole in the BV421-A channel and FSC-area parameters were used to exclude dead cells. The identified live singlet population was then analyzed and sorted for double positive eGFP- and mScarlet-expressing cells. Single cells were sorted into 96-well plates (3596; Costar) prefilled with 100 μl of media using FITC-A and PE-Texas Red-A settings. The selected cells were then incubated at 37 °C in a humidified environment of 5% CO₂ for a minimal of 6 weeks, changing media every 2 weeks.

Negi H., Osei-Amponsa V., Ibrahim B., Evans C.N., Sullenberger C., Loncarek J., Chari R, & Walters K.J. (2023). An engineered cell line with a hRpn1-attached handle to isolate proteasomes. The Journal of Biological Chemistry, 299(8), 104948.

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Co-culture of ES-2-WT and RAW 264.7 Cells for Tumor Xenograft Model

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ES-2-WT cells were co-cultured with RAW 264.7 murine macrophages in RPMI medium with 10% fetal bovine serum and 5% Penicillin/Streptomycin, and initial proportion of 3:1 of ES-2-WT cells and RAW 264.7, respectively. Nude mice were injected with ES2-WT cells (3×10⁶ cell per 100 μL in PBS). After 10 days, the control group received PBS injection (100 uL), and the treatment group received injections with GFP mRNA LNP formulation (2ug mRNA/mouse in 100uL PBS). Ascitic fluid was collected by IP lavage. Both in vitro and in vivo samples were fixed with 1:1 acetone/methanol solution pre-cooled to −20 °C and blocked with 1:1 mixture of TruStain FcX Fc receptor blocking solutions (human and murine, Biolegend, San Diego, CA, USA) for 30 minutes at room temperature, followed by conjugated antibodies staining for HLA-ABC (HLA-ABC Monoclonal Antibody (W6/32), PE eBioscience, Thermofisher Scientific, Waltham, MA, USA) and GFP (APC Anti-GFP antibody, Abcam, Cambridge, UK). Flow cytometry was performed on BD FACSymphony S6 cell sorter (BD, Franklin lakes, NJ, USA) using laser excitation wavelength of 561 nm and 628 nm for HLA-ABC and GFP detection, respectively. Data was analyzed using FlowJo (FlowJo LLC, Ashland, OR, USA).

Korzun T., Moses A.S., Kim J., Patel S., Schumann C., Levasseur P.R., Diba P., Olson B., De Oliveira Rebola K.G., Norgard M., Park Y., Demessie A.A., Eygeris Y., Grigoriev V., Sundaram S., Pejovic T., Brody J.R., Taratula O.R., Zhu X., Sahay G., Marks D.L, & Taratula O. (2022). Nanoparticle-Based Follistatin Messenger RNA Therapy for Reprogramming Metastatic Ovarian Cancer and Ameliorating Cancer-Associated Cachexia. Small (Weinheim an der Bergstrasse, Germany), 18(44), e2204436.

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SARS-CoV-2 Spike-specific Memory T Cell Analysis

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Cryopreserved PBMVs from 6 BNT162b2 vaccinated donors were incubated in 1 ml RPMI 1640 medium containing 50 U/ml benzonase nuclease (Millipore, Darmstadt, Germany), 10% fetal bovine serum, and penicillin–streptomycin for 2 h. Next, cells were incubated in 200 µl medium with or without peptides (17-mers overlapping by 11 residues) corresponding to the full-length SARS-CoV-2 spike WT or Omicron strain, at a final concentration of 2 µg/ml of each peptide, for 4 h in presence of anti-4-1BB (4B4-1). The cells were then stained using the LIVE/DEAD Fixable Violet Dead Cell Stain Kit (Thermo Fisher Scientific), and stained with anti-CD3 (SP34-2), anti-CD8 (RPA-T8), anti-CD4 (L200), anti-CD45RO (UCHL1), anti-CD27 (1A4CD27), and anti-CD69 (FN50) antibodies. Antibodies of anti-CD16 (3G8), anti-TCRγ/δ (B1), anti-CD14 (M5E2), anti-CD56 (NCAM16.2), and anti-CD20 (2H7) were additional stained in the dump channels and excluded from analysis. The 200 cells gated to dump^-CD3⁺CD8⁺CD69⁺4-1BB⁺ memory T cells based on CD27 and CD45RO expression were sorted using BD FACSymphony S6 cell sorter (BD Biosciences).

Nogimori T., Suzuki K., Masuta Y., Washizaki A., Yagoto M., Ikeda M., Katayama Y., Kanda H., Takada M., Minami S., Kobayashi T., Takahama S., Yoshioka Y, & Yamamoto T. (2023). Functional changes in cytotoxic CD8+ T-cell cross-reactivity against the SARS-CoV-2 Omicron variant after mRNA vaccination. Frontiers in Immunology, 13, 1081047.

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SARS-CoV-2 Replicon Transfection and IFN-γ Treatment

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293 T cells cultured in T25 flasks were transfected with 10 μg SARS-CoV2 (Wuhan) ∆N-EGFP replicon DNA per flask, using Lipofectamine^TM 3000 (Invitrogen) transfection reagent. Two days after transfection, the cells were treated with or without IFN-γ (500 U/ml) for 24 h. Treated and untreated GFP⁺ cells were collected by sorting using the BD FACSymphony™ S6 Cell Sorter (BD Biosciences).

Khan D., Terenzi F., Liu G., Ghosh P.K., Ye F., Nguyen K., China A., Ramachandiran I., Chakraborty S., Stefan J., Khan K., Vasu K., Dong F., Willard B., Karn J., Gack M.U, & Fox P.L. (2023). A viral pan-end RNA element and host complex define a SARS-CoV-2 regulon. Nature Communications, 14, 3385.

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Multiparametric Immune Cell Profiling

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For the surface stain, 1x10⁶ PBMCs were resuspended in 100 ul PBS with 2% FBS (FACS buffer) and incubated with BD human FC block (BD Biosciences, San Diego, CA) for 10 min at room temperature (RT). Without washing, fluorescently-labeled chemokine receptor antibodies were added to cells and incubated at 37°C in the dark for 10 min. Antibody mix containing the rest of the surface antibodies were then added directly to cells and incubated for 20 min at RT in the dark. Following surface staining, cells were washed once with FACS buffer and resuspended in 100 uL BD fixation/permeabilization solution (BD Biosciences, San Diego, CA) and incubated at 4°C for 45 min in the dark. Cells were then washed twice with permeabilization buffer and stained with intracellular and intranuclear antibodies for 20 min at 4°C in the dark. After staining, cells were washed once with permeabilization buffer and resuspended in FACS buffer. All samples were acquired on a BD FACSymphony S6 cell sorter. A list of antibodies used in this panel can be found in Table S3.

Rydyznski Moderbacher C., Ramirez S.I., Dan J.M., Grifoni A., Hastie K.M., Weiskopf D., Belanger S., Abbott R.K., Kim C., Choi J., Kato Y., Crotty E.G., Kim C., Rawlings S.A., Mateus J., Tse L.P., Frazier A., Baric R., Peters B., Greenbaum J., Ollmann Saphire E., Smith D.M., Sette A, & Crotty S. (2020). Antigen-Specific Adaptive Immunity to SARS-CoV-2 in Acute COVID-19 and Associations with Age and Disease Severity. Cell, 183(4), 996-1012.e19.

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