Imagestream x mark 2

Name: Imagestream x mark 2
Brand: Merck Group
SKU: GCfiCZIBPBHhf-iFHF7k
Availability: InStock

Manufactured by Merck Group

80 citations

Sourced in United States, Belgium, Germany

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The ImageStream X Mark II is a multi-spectral imaging flow cytometer developed by Merck Group. It combines the capabilities of flow cytometry and microscopy to capture high-resolution images of individual cells or particles while they flow through the system. The ImageStream X Mark II allows for the simultaneous analysis of morphology, location, and fluorescence of cells or particles.

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80 protocols using «imagestream x mark 2»

Yeast-Bacteria Adhesion Assay by Flow Cytometry

2024

All samples were measured on a BD Accuri™ C6 Plus flow cytometer (BD Bioscience) equipped with a laser exciting at a wavelength of 488 nm. Before each experiment, the calibration of the flow cytometer was assessed with calibration beads (BDTM CS&T RUO Beads; BD Biosciences). Acquisition parameters were set at a flow rate of 14 µl/min and 10 µm core size with a threshold at 10,000 on SSC-Height signal. A first gate was applied to select the population corresponding to yeast cells which was based on the dot plot of the forward scatter channel (FSC-Area) versus side scatter channel (SSC-Area) that excluded free bacteria. In addition, based on the FSC-Area vs FSC-Height dot plot, single yeast (singlets) cells with similar cell size and granularity were selected by applying a second gate (Fig. S1). The Dendra2 green fluorescence emissions of the bacteria were analyzed with the FL1 channel (533/30 nm). Finally, the adhesion index (A_i) was computed as the amount of yeast singlets with bound green, fluorescent bacteria (FL1 channel) divided by the total number of yeast singlets × 100. Data acquisition was set at 100,000 events per sample and after applying the two gates at least 70,000 events of single yeast cells were analyzed per sample. Data were analyzed with FlowJo v10 software (BD Bioscience) and same gates were applied to all the data, to ensure that it included the entire population especially in the case of morphological variations between strains. No variations in cell population between yeast strains used in this study were seen, except for the strain gas1Δ.
Imaging flow cytometry of mix of bacteria (5 × 10⁹ cell/ml) and yeast (10⁸ cell/ml) initially preincubated for 90 min at 37 °C was acquired on the ImageStreamX Mark II (Amnis-EMD Millipore). Yeasts were selected on the basis of their size on dot plot Area and Aspect ratio on the brightfield. For each experiment, 1000 yeast per sample were acquired (ensuring a sufficient number of events remaining for statistically robust analysis) at 60X magnification. Image analysis was completed using image-based algorithms in the ImageStream Data Exploration and Analysis Software (IDEAS_6.2.64.0, EMD Millipore). The number of fluorescent bacteria puncta per yeast was assessed with the feature “Spot Counting”. A mask based on GFP staining was employed (SpotCount_Peak [M02, Ch02, Bright, 18.95]_4) to visualize bacteria. The same mask and feature were automatically applied for each condition.

Schiavone M., Dagkesamanskaya A., Vieu P.G., Duperray M., Duplan-Eche V, & François J.M. (2024). A flow cytometry method for quantitative measurement and molecular investigation of the adhesion of bacteria to yeast cells. Scientific Reports, 14, 20935.

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Multiparametric Imaging of TIL Activation

2024

For Imagestream analysis, live TILs at 1 × 10⁷ cells per ml were run at 100 cells per second on the ImageStreamX MarkII (Merck Millipore). TILs were stained with relevant antibodies for CD4, CD8, IL-2Rα, IL-2Rβ, IL-2Rγ_c and DAPI. Single stained cells were used as compensation controls. Images were captured at 60× magnification. Data were analysed using the ImageStream Data Analysis and Exploration Software (IDEAS). Colocalization was calculated based on Bright Detail Similarity score, a log-transformed Pearson’s correlation coefficient computed by Amnis.

Morotti M., Grimm A.J., Hope H.C., Arnaud M., Desbuisson M., Rayroux N., Barras D., Masid M., Murgues B., Chap B.S., Ongaro M., Rota I.A., Ronet C., Minasyan A., Chiffelle J., Lacher S.B., Bobisse S., Murgues C., Ghisoni E., Ouchen K., Bou Mjahed R., Benedetti F., Abdellaoui N., Turrini R., Gannon P.O., Zaman K., Mathevet P., Lelievre L., Crespo I., Conrad M., Verdeil G., Kandalaft L.E., Dagher J., Corria-Osorio J., Doucey M.A., Ho P.C., Harari A., Vannini N., Böttcher J.P., Dangaj Laniti D, & Coukos G. (2024). PGE2 inhibits TIL expansion by disrupting IL-2 signalling and mitochondrial function. Nature, 629(8011), 426-434.

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Corresponding organizations : Ludwig Cancer Research, University of Lausanne, Technical University of Munich, Helmholtz Zentrum München

Flow Cytometry Characterization of SARS-CoV-2

2024

Flow cytometry was carried out under the recommendation of MIFlowCyt‐EV.^[⁷⁸ (link)
^] SARS‐CoV‐2 virions were stained with antibodies targeting the spike glycoprotein and molecular beacons targeting the nucleocapsid‐encoding RNA (Table S10). MLV virions were used as an isotype control and PBS as a negative control. The samples were stained following the same protocol. To confirm SARS‐CoV‐2 signals, samples were treated with 0.5% Triton X‐100 for 30 min at room temperature to lyse the stained SARS‐CoV‐2, according to the reported protocol.^[⁷⁹ (link)
^] Data acquisition was performed on an ImageStream^X Mark II imaging flow cytometer (MilliporeSigma, Burlington, MA, USA) using INSPIRE software. All samples were measured following the same acquisition protocol. Calcium and magnesium‐free PBS was used as sheath fluid. In brief, a flow speed of 44 mm ⁻¹s and a 7 µm diameter core size was utilized. The camera was set to the highest bin mode resolution, a sensitivity of 32 for all channels, a gain of 1, and a gain of 1 on the second camera. All acquisition was performed at a 60× magnification. The 561‐nm laser at channel 3 was set to a power of 200 mW, the 642‐nm laser at channel 11 was set to a power of 150 mW, and the 785‐nm laser for exciting side scatter (SSC) at channel 6 was set to a power 70 mW, where channel 1 was used for bright field with an LED intensity at 52.83 mW. The fluorescence signals of spike glycoprotein and molecular beacons were collected in channel 11 and channel 3, respectively. All sample events were acquired for 3 min. Data analysis was performed using Amnis IDEAS software (version 6.2.64.0). Gating was performed based on unstained, buffer‐stained, and stained samples.

Nguyen K.T., Rima X.Y., Nguyen L.T., Wang X., Kwak K.J., Yoon M.J., Li H., Chiang C., Doon‐Ralls J., Scherler K., Fallen S., Godfrey S.L., Wallick J.A., Magaña S.M., Palmer A.F., Lee I., Nunn C.C., Reeves K.M., Kaplan H.G., Goldman J.D., Heath J.R., Wang K., Pancholi P., Lee L.J, & Reátegui E. (2024). Integrated Antigenic and Nucleic Acid Detection in Single Virions and Extracellular Vesicles with Viral Content. Advanced Healthcare Materials, 14(3), 2400622.

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Imaging Flow Cytometry Colocalization Analysis

2024

Imaging flow cytometry was performed exactly as described before with an ImageStream X Mark II (Merck Millipore, Burlington, MA, USA) one-camera system with 351, 488, 562, 658, and 732 nm lasers [31 (link)]. Analyte-positive cells were discriminated based on controls. The Bright Detail Similarity R3 feature was used to quantify the degree of colocalization in double-positive cells only [31 (link)].

Ploeger L., Kaleja P., Tholey A., Lettau M, & Janssen O. (2024). Analysis of Cytotoxic Granules and Constitutively Produced Extracellular Vesicles from Large Granular Lymphocytic Leukemia Cell Lines. Cells, 13(16), 1310.

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Annexin V-FITC and PI Apoptosis Assay

2024

The apoptosis of cells following NP exposure was identified through the utilization of fluorescent probes, namely Annexin V-FITC and propidium iodide (PI) (Solarbio, Beijing, China). Subsequently, the stained cells (3 × 10⁶ cells/well) were taken and washed twice with PBS, before being resuspended in 500 μL of binding solution, with the cell concentration maintained at 1 × 10⁵ to 5 × 10⁵ cells per mL. Annexin V-FITC and PI were added to the cytosol at a ratio of 5 μL each, mixed thoroughly, and incubated for 15 min at room temperature in the dark. To prevent cross-talk between fluorescence channels, single-stained samples of experimental groups were prepared for fluorescence compensation. The apoptosis rate of earthworm immune cells was determined using a flow cytometer (ImageStreamX MarkII, Merck, Darmstadt, Germany) with an excitation wavelength of 488 nm, and the data were processed with FlowJo software (v10.7.2).

Shi H., Wang Y., Li X., Wang X., Qi Y., Hu S, & Liu R. (2024). Polystyrene Nanoplastics Elicit Multiple Responses in Immune Cells of the Eisenia fetida (Savigny, 1826). Toxics, 13(1), 18.

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Top 5 most cited protocols using «imagestream x mark 2»

Quantitative Imaging of Influenza Viral Proteins

Cited 3 times

Stored samples were centrifuged for 10 min at 300×g and 4 °C and the supernatant was discarded. Then, cells were washed in 4 mL fluorescence-activated cell sorting (FACS) buffer (PBS, 2 % (w/v) glycine, 0.1 % (w/v) bovine serum albumin (BSA)) and centrifuged as before. Subsequently, cells were blocked in 150 μL FACS buffer containing 1.1 % (w/v) BSA for 1 h on ice. After centrifugation for 10 min at 300×g and 4 °C, cell pellets were resuspended in 100 μL antibody solution. All antibody incubations were performed at 37 °C for 1 h in the dark. The monoclonal mouse anti-NP antibody mAb61A5 (a kind gift from Fumitaka Momose) was used at a dilution of 1:500. This antibody preferentially binds to NP in the conformation inherent to the vRNP complex (Momose et al. 2007 (link)). Following incubation, the cells were washed three times with FACS buffer. Secondary antibody staining was performed using Alexa Fluor 647-conjugated polyclonal goat anti-mouse antibody (LifeTechnologies, #A21235) at a dilution of 1:500. Subsequently, cells were washed three times with wash buffer and 4′,6-diamidino-2-phenylindole (DAPI) was used for nuclear staining.
The immunostaining of M1 was performed using a FITC-conjugated monoclonal mouse anti-M1 antibody (AbD serotec, #MCA401FX) at a dilution of 1:100. After incubation and three washing steps, cells were resuspended in 40 μL of wash buffer. RNA degradation was conducted by adding 5 μL PureLink™ RNase A (20 mg/mL, life technologies). For nuclear staining, 0.5 μL of 7-AAD (Millipore) were added followed by an incubation for 30 min at room temperature in the dark.
Using the ImageStream X Mark II (Amnis, EMD Millipore) 10,000 single cells per sample (debris and cell doublets were excluded) were analyzed using ×40 or ×60 objective lenses. For infection experiments at low MOI, up to 300,000 single cells were measured. The 375 and 642 nm lasers were utilized for the excitation of the DAPI- and vRNP-stained samples. Channels 1 (CH1) and 5 (CH5) were acquired along with the bright field (BF) imagery on channel 6 (CH6). For the M1- and 7-AAD-stained samples, the 488 and 642 nm lasers were utilized for excitation and signal acquisition was conducted in channels 2 (CH2) and 5 (CH5) along with the BF imagery on channel 1 (CH1). Before acquisition, the laser power was adjusted to yield a “raw max pixel” feature value between 200 and 1500 of the single-stained positive controls. One thousand cells of these samples were acquired for compensation with the respective compensation settings.

Frensing T., Kupke S.Y., Bachmann M., Fritzsche S., Gallo-Ramirez L.E, & Reichl U. (2016). Influenza virus intracellular replication dynamics, release kinetics, and particle morphology during propagation in MDCK cells. Applied Microbiology and Biotechnology, 100, 7181-7192.

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Corresponding organizations : Max Planck Institute for Dynamics of Complex Technical Systems, Otto-von-Guericke University Magdeburg

Multiparametric Characterization of Dendritic Cells

Cited 1 time

mDCs were stained with FITC‐labeled anti‐CD40 (5C3: BD Biosciences), FITC‐labeled anti‐CD80 (2D10: BioLegend), FITC‐labeled anti‐CD83 (HB15a: Beckman Coulter), FITC‐labeled anti‐CD86 (2331: BD Biosciences), FITC‐labeled anti‐CD274 (programmed death ligand 1) (MIH2: BioLegend), or PE‐labeled anti‐HLA‐DR (TU36: BD Biosciences) after 24 h of culture and with PE‐labeled anti‐OX40L (ANC10G1: Ancell) after 48 h of culture, then analyzed using a FACS Calibur (BD Biosciences). Isotype‐matched mAbs (R&D Systems) were used as negative controls. Viable cells were counted in triplicate using trypan‐blue exclusion to identify dead cells and were also evaluated as annexin V‐negative fractions using an annexin V‐FITC Apoptosis Detection kit (Sigma–Aldrich) after 24 h of culture. The production of CCL17 in the culture supernatants after 24 h was determined by ELISA (R&D Systems). To evaluate intracellular phosphorylated (p) STAT6 and NF‐κB‐p50, mDCs were preliminarily cultured for 6 h with medium alone to induce upregulation of the TSLP receptor, and then stimulated with TSLP ± statins for 3 h, after which the cells were immediately fixed using a Cytofix/Cytoperm kit (BD biosciences) and stained using Alexa 488‐labeled anti‐NF‐κB‐p50 (4D1: BioLegend) and Alexa 488‐labeled or PE‐labeled anti‐phospho‐stat6 (18/P‐Stat6: BD Biosciences). The stained cells were then analyzed using a FACSCalibur and visualized by ImageStream X mark II (Merck Millipore). In some experiments, PI was used for nuclei staining.

Inagaki‐Katashiba N., Ito T., Inaba M., Azuma Y., Tanaka A., Phan V., Kibata K., Satake A, & Nomura S. (2019). Statins can suppress DC‐mediated Th2 responses through the repression of OX40‐ligand and CCL17 expression. European Journal of Immunology, 49(11), 2051-2062.

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Corresponding organizations : Kansai Medical University

Phagocytosis Assay with Macrophages and MOLM-13 Cells

Phagocytosis assay was performed as described previously [53 ]. Briefly, isolated monocytes were stained with PKH67 (Sigma-Aldrich) according to the manufacturer's instructions and differentiated to macrophages by 20 ng/ml Macrophage-Colony Stimulator Factor (M-CSF) (R&D Systems) in X-VIVO 10 medium (Lonza) supplemented with 10% autologous serum. MOLM-13 cells were stained with PKH26 (Sigma-Aldrich) following the manufacturer's instructions and incubated in a 1:2 E:T ratio with licMABs or mAb concentrations ranging from 0.01 nM to 100 nM for 2 h. Polybead^® Carboxylate Red-Dyed Microspheres of 6 μm (Polysciences) were used as a positive control and incubation either at 4°C or at 37°C in the presence of 10 μM Cytochlasin D (Sigma-Aldrich) served as a negative control. Cells were harvested, measured by imaging flow cytometry using an ImageStream^®X Mark II instrument (Merck Millipore, Billerica, Massachusetts, USA) and analyzed with IDEAS^® and INSPIRE^® Software (Merck Millipore, Billerica, Massachusetts, USA). The maximum phagocytosis value was set to 100% and all data points were normalized accordingly. Mean values and standard errors of triplicates were calculated and plotted.

Ponce L.P., Fenn N.C., Moritz N., Krupka C., Kozik J.H., Lauber K., Subklewe M, & Hopfner K.P. (2017). SIRPα-antibody fusion proteins stimulate phagocytosis and promote elimination of acute myeloid leukemia cells. Oncotarget, 8(7), 11284-11301.

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Corresponding organizations : Ludwig-Maximilians-Universität München, Helmholtz Zentrum München

Multiparametric cell analysis protocol

For flow and imaging cytometry cells were washed twice with 2.5mmol EDTA in PBS, detached with trypsin-EDTA solution (0.05%) (Thermo, Reinach, Switzerland) and put through a 35μm cell strainer (Falcon, Corning, USA) after resuspension. The fixable viability dye eFluor® 660 (e Bioscience, Hatfield, UK) was used for viability analysis in accordance with the manufacturer’s instructions. For cell analysis, BD FACS Canto II (BD) and Image Stream X Mark II (Merck, Millipore, Schaffhausen, Switzerland) were used. Doublets were excluded with the aid of a pulse geometry gate (FS-H x FS-A) in the flow cytometer and microscopically in the Image Stream analysis. Dead cells were excluded upon staining. For the flow cytometry analysis, the FSC-A voltage values of the first 500 cells in each experiment were analyzed and normalized to normoxia. For image stream analysis, cell size for every measured cell was determined. ANOVA and Bonferroni correction was used for comparison of the different conditions.

Restin T., Kajdi M.E., Schläpfer M., Roth Z’graggen B., Booy C., Dumrese C, & Beck-Schimmer B. (2017). Sevoflurane protects rat brain endothelial barrier structure and function after hypoxia-reoxygenation injury. PLoS ONE, 12(10), e0184973.

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Corresponding organizations : Swiss Integrative Center for Human Health, University of Zurich, University Hospital of Zurich, University of Illinois Chicago

Imaging Flow Cytometry Analysis of Influenza Infection

The relative amount of infected cells, the percentage of vRNP in the cell nucleus and the percentage of apoptotic cells were determined using imaging flow cytometry, based on previously established assays [97 (link)]. Cell fixation was carried out using 1 mL of infected MDCK cells, which were mixed with paraformaldehyde to a final concentration of 2% and incubated at 4 °C for 30 min. Thereafter, the cells were washed with PBS (300× g, 10 min, 4 °C), added to 5 mL 70% ethanol (−20 °C) and stored at −20 °C. For staining, fixed cells in ethanol were centrifuged (300× g, 10 min, 4 °C) to remove the storage solution. The cell pellet was washed twice with FACS-buffer (PBS containing 0.1% BSA and 2% glycine) and blocked in PBS containing 1% BSA (30 min, 37 °C). vRNP positive cells were stained with a monoclonal mouse anti-NP antibody mAb61A5 [98 (link)] as a primary antibody, and Alexa Fluor 647-conjugated goat anti-mouse pAb (#A21235, Thermo Scientific, Waltham, MA, USA,) as a secondary antibody. All antibodies were incubated for 60 min at 37 °C in FACS-buffer. Between each incubation step, cells were washed twice with FACS-buffer (300× g, 10 min, 4 °C). Before analysis, nucleic DNA was stained with DAPI (50 mg/L, #6843.2, Carl Roth, Karlsruhe, Germany). Ten thousand single cells were analyzed with an ImageStream X Mark II (#100220, Merck, Darmstadt, Germany) using a 60× objective lens. Image processing was carried out with the IDEAS software (version 6.1). The vRNP-positive cells were considered infected and nucleic condensation and fragmentation were used as signs of apoptosis.

Ramos J.R., Bissinger T., Genzel Y, & Reichl U. (2022). Impact of Influenza A Virus Infection on Growth and Metabolism of Suspension MDCK Cells Using a Dynamic Model. Metabolites, 12(3), 239.

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Corresponding organizations : Max Planck Institute for Dynamics of Complex Technical Systems, Otto-von-Guericke University Magdeburg

Spelling variants (same manufacturer)

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