Cytoflex s platform

Manufactured by Beckman Coulter

Sourced in United States

The CytoFLEX S platform is a flow cytometer designed for multi-color analysis of cells and particles. It features a compact, benchtop design and utilizes solid-state lasers to generate multiple excitation wavelengths. The CytoFLEX S platform is capable of detecting and measuring various parameters, including size, granularity, and the expression of fluorescently-labeled markers on cells or other biological entities.

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Lab products found in correlation

Ultracomp beads, by Thermo Fisher Scientific (2 mentions) Fcr blocking reagent, by Miltenyi Biotec (2 mentions) Mhc class 2, by Miltenyi Biotec (1 mentions) Mhc class 1, by Thermo Fisher Scientific (1 mentions) Hepes, by Thermo Fisher Scientific (1 mentions) Dispase 2, by Merck Group (1 mentions) Live dead fixable violet dead cell stain sampler kit, by Thermo Fisher Scientific (1 mentions) Collagenase 2, by Thermo Fisher Scientific (1 mentions) Brefeldin a, by Merck Group (1 mentions) Ionomycin, by Merck Group (1 mentions) Phorbol myristate acetate (pma), by Merck Group (1 mentions) Cytofix cytoperm, by BD (1 mentions) Faststart essential dna green master reagent, by Roche (1 mentions) Anti rab5, by Santa Cruz Biotechnology (1 mentions) Sc 46692, by Santa Cruz Biotechnology (1 mentions) Anti tubulin, by Merck Group (1 mentions) Lightcycler 96 real time pcr system, by Roche (1 mentions) Dneasy tissue kit, by Qiagen (1 mentions) Anti calreticulin, by Thermo Fisher Scientific (1 mentions) Rpmi medium, by Avantor (1 mentions)

6 protocols using cytoflex s platform

Detailed Flow Cytometry Protocol

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Flow cytometric measurements were performed using the CytoFLEX S platform (Beckman Coulter Genomics, Brea, CA, USA). For extracellular staining of T cells, isolated splenocytes were incubated for 25 min with FcR blocking reagent (Miltenyi Biotec, Bergisch Gladbach, Germany); washed in PBS; incubated for 1 h at room temperature with CD3, CD4, and CD8 (Miltenyi Biotec) staining antibodies or H-2kb OVA Tetramer-SIINFEKL (MBL International, Woburn, MA, USA); washed again; and processed according to the manufacturer’s protocols.
Invitro-generated and activated DCs were washed in PBS, incubated for 30 min at room temperature with the 7-AAD viability dye (Sigma Life Sciences, Merck, Darmstadt, Germany), CD11c, CD80 (BioLegend, San Diego, CA, USA), CD86 (BD Biosciences, Franklin Lakes, NJ, USA), MHC class I (Invitrogen, Thermo Fisher Scientific), MHC class II (Miltenyi Biotec) DC lineage and activation marker antibodies, washed again, and processed according to the manufacturer’s protocols. The compensation was performed based on staining results from UltraComp beads (Thermo Fisher Scientific). All flow cytometry results were analyzed using Flow Jo (Ashland, OR, USA) software.

Marek J., Hanesch L., Krabbe T., El Khawanky N., Heidegger S, & Altomonte J. (2023). Oncolytic virotherapy with chimeric VSV-NDV synergistically supports RIG-I-dependent checkpoint inhibitor immunotherapy. Molecular Therapy Oncolytics, 30, 117-131.

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Isolation and Analysis of Cytotoxic T Cells from Prostate Cancer

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CD8⁺CXCR6⁺T and CD8⁺CXCR6^‐T were enriched with flow cytometry. Prostates were minced and digested at 37 °C for 45 min in media containing DMEM supplemented with 1% FBS, 1% glutamine, 1% non‐essential amino acids, 1% sodium pyruvate, 1% penicillin/streptomycin, 10 mM HEPES (Invitrogen Life Technologies), 0.2% collagenase II (Gibco, 17101015), 2.5 U mL⁻¹ Dispase II (Sigma‐Aldrich, D4693). Intracellular staining for GZMB and PRF1 were ducted using BD Cytofix/Cytoperm and BD Perm/Wash buffers (BD Biosciences, 554714) according to the manufacture's recommendations. To detect intracellular GZMB and PRF1, the enriched cells isolated from prostate cancer tissues were incubated with 50 ng mL⁻¹ PMA (Sigma‐Aldrich), 500 ng mL⁻¹ ionomycin (Sigma‐Aldrich), and 10 mg mL⁻¹ brefeldin A (Sigma‐Aldrich) for 4 h at 37 °C and stained as described earlier. Samples were analyzed on a CytoFLEX S platform (Beckman Coulter) and FlowJo V10.4 software. The antibodies used were as follows: Alexa 488 conjugated anti‐human CD8 (Cat. No. 557704; BD Biosciences), PE anti‐human CD186 (CXCR6) (Cat. No. 356004; Biolegend), and BV421 anti‐human GZMB (BD Biosciences; 563389), BV421 anti‐human PRF1(BD Biosciences; 563393), and LIVE/DEAD Fixable Violet Dead Cell Stain Sampler Kit (ThermoFisher, L34964).

Bian X., Wang W., Abudurexiti M., Zhang X., Ma W., Shi G., Du L., Xu M., Wang X., Tan C., Sun H., He X., Zhang C., Zhu Y., Zhang M., Ye D, & Wang J. (2024). Integration Analysis of Single‐Cell Multi‐Omics Reveals Prostate Cancer Heterogeneity. Advanced Science, 11(18), 2305724.

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Flow Cytometric Analysis of Immune Markers

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Flow cytometric measurements were performed using the CytoFLEX S platform (Beckman Coulter, Brea, CA, USA). If not stated otherwise, samples were incubated for 25 min with FcR blocking reagent (Miltenyi Biotech, Bergisch Gladbach, Germany), washed in PBS, incubated for 1 h at room temperature with Viobility Fixable Dye, CD8, CD4 (Miltenyi Biotech, Bergisch Gladbach, Germany), PD1, IFNγ, and TNFα (BD Biosciences, Franklin Lakes, NJ, USA) staining antibodies or SIINFEKL(OVA)-/ (VSV-NP)-specific tetramers (MBL International, Woburn, MA), washed again and processed according to the manufacturers’ protocols. The compensation was performed based on staining results from UltraComp beads (Thermo Fisher Scientific, Waltham, MA, USA). The results were analyzed using Flow Jo (Ashland, OR, USA) software.

Krabbe T., Marek J., Groll T., Steiger K., Schmid R.M., Krackhardt A.M, & Altomonte J. (2021). Adoptive T Cell Therapy Is Complemented by Oncolytic Virotherapy with Fusogenic VSV-NDV in Combination Treatment of Murine Melanoma. Cancers, 13(5), 1044.

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Quantitative Analysis of Viral Transduction

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HeLa cells, cultured in DMEM (10% FBS 1% Pen/Strep), were plated in 6‐well plates. 24 h later, cells were counted and transduced with vector particles at a particle per cell ratio of 1,000. 24 h later, cells were harvested by extensive trypsin treatment and PBS washing steps to remove any membrane bound vector particles. Cells were counted and 3–5 × 10⁵ cells were used for subcellular fractionation, while 10⁵ cells were analysed by flow cytometry (CytoFlex S platform, Beckman Coulter) to determine transduction efficiency. Subcellular fractionation was performed as previously described (Rossi et al, 2019). Membrane, cytosolic and nuclear fractions were collected. Purity of fractions was confirmed by Western blot using anti‐Rab 5 (1:100, sc46692, Santa Cruz), anti‐Tubulin (1:5,000, T5198, Sigma Aldrich), anti‐Lamin B1 (1:5,000, 16048, Abcam) and anti‐Calreticulin (1:100, PA3‐900, Affinity BioReagents) antibodies. Fractions were subjected to DNA isolation (Qiagen, DNeasy Tissue kit) followed by qPCR analysis (FastStart essential DNA green master reagent, Roche) using CMV promoter‐specific primers on the LightCycler 96 real‐time PCR system (Roche). The specificity of target DNA amplification was confirmed by melting‐curve analysis. All samples were run in technical duplicates.

Pavlou M., Schön C., Occelli L.M., Rossi A., Meumann N., Boyd R.F., Bartoe J.T., Siedlecki J., Gerhardt M.J., Babutzka S., Bogedein J., Wagner J.E., Priglinger S.G., Biel M., Petersen‐Jones S.M., Büning H, & Michalakis S. (2021). Novel AAV capsids for intravitreal gene therapy of photoreceptor disorders. EMBO Molecular Medicine, 13(4), e13392.

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TRPV1-Mediated CD69 Externalization in Eosinophils

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To assess CD69 externalization after TRPV1 activation, purified eosinophils were stimulated with 0.1, 1, 10, or 100 µM capsaicin (Merck, Darmstadt, Germany), with IL-3 (10 ng/mL) (PeproTech, Cranbury, NJ, USA) and GM-CSF (10 ng/mL) (BioLegend, Amsterdam, the Netherlands) as positive controls, for 24 h at 37 °C and 5% CO₂ in RPMI medium (containing 10% FCS and 1% PenStrep) (VWR International, Leuven, Belgium). To analyze CD69 and TRPV1 expression, eosinophils were stained with CD69-APC (Miltenyi Biotec, Bergisch Gladbach, Germany) and TRPV1-PE (Biozol, Eching, Germany) antibodies. FMO controls were stained without the CD69-APC or TRPV1-PE antibody, respectively. To exclude unspecific binding, cells were also stained with a PE-Rabbit Isotype Control (ab37407, Abcam, Cambridge, UK) or REA APC isotype control (Miltenyi Biotec, Bergisch Gladbach, Germany). Measurement was performed after 10 min of incubation in the dark on the CytoFlexS platform (Beckman Coulter, Brea, CA, USA). CD193-FITC, CD69-APC, CD15-PB, and CD16-APC-A750 were compensated using the MACS Comp Bead Kit anti-REA (Miltenyi Biotec, Bergisch Gladbach, Germany) for the REA antibodies and the MACS Comp Bead Kit anti-mouse Igκ (Miltenyi Biotec, Bergisch Gladbach, Germany) for the remaining antibodies, according to the manufacturer’s protocol.

Weihrauch T., Gray N., Wiebe D., Schmelz M., Limberg M.M, & Raap U. (2024). TRPV1 Channel in Human Eosinophils: Functional Expression and Inflammatory Modulation. International Journal of Molecular Sciences, 25(3), 1922.

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Plasma miRNA Detection via Nanoscale Flow Cytometry

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Blood samples were drawn from the veins of participants using the BD Vacutainer EDTA blood collection tube (BD Biosciences, CA, USA). The blood was centrifuged at 4°C and 2000g for 15 min within 3 hours since collection. The plasma was transferred to a new tube and stored at −80°C for subsequent experiments. Right before the miRNA detection, the plasma was thawed on ice and centrifuged at 4°C and 12,000g for 30 min. Five microliters of plasma from the supernatant was mixed with 50 μl of miR-44438–NCMB at a concentration of 10 nM and incubated at 37°C for 8 hours. After the incubation, we added 150 μl of phosphate-buffered saline (PBS; pH 7.4) to the mixture for a 1:4 dilution and analyzed it using the nanoscale flow cytometry with the Cytoflex S platform (Beckman Coulter, Milano, Italy). To identify small-sized EVs, we used the side scatter mode with a violet excitation laser, which we referred to as violet side scatter (VSSC). Because the VSSC mode introduced a lot of background noise, we used the volumetric measurement to obtain the fluorescence-positive event concentrations (events per milliliter) instead of the ratio of positive to total events. The Apogee Mix (catalog no. 1493) containing 110- and 500-nm green fluorescent polystyrene beads was used for the daily standardization.

Yu Z., Zheng Y., Cai H., Li S., Liu G., Kou W., Yang C., Cao S., Chen L., Liu X., Wan Z., Zhang N., Li X., Cui G., Chang Y., Huang Y., Lv H, & Feng T. (2024). Molecular beacon–based detection of circulating microRNA-containing extracellular vesicle as an α-synucleinopathy biomarker. Science Advances, 10(20), eadl6442.

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