Nucleated cells were collected from whole blood via red blood cell sedimentation using HetaSep solution (STEMCELL Technologies, Cat# 07906). Neutrophils were isolated as previously described (plasma centrifuged, PBMCs isolated and collected, and neutrophils isolated from remaining red blood cell pellet). Isolated neutrophils and nucleated cells were centrifuged at 300g for 5 minutes at room temperature with the brakes off. The cells were then resuspended in 50 μl of FACS buffer (0.5% BSA in PBS) and then fixed in 1% paraformaldehyde in PBS. All fixed cells were then stained with CD66b (1:200 dilution) (Biolegend, Cat# 305103), CD14 (1:20 dilution) (Biolegend, Cat# 325615), and DRAQ5 (1:2000 dilution) (Cell Signaling Technology, Cat# 4084S). Data was obtained through the Amnis ImageStreamX Mark II imaging flow cytometer and INSPIRE software (EMD Millipore, Billerica, MA, USA). The accompanying IDEAS software (EMD Millipore) was used to perform data analysis.
Imagestream x mark 2 imaging flow cytometer
Manufactured by Merck Group
Sourced in United States, Germany
The ImageStream®X Mark II Imaging Flow Cytometer is a lab equipment product manufactured by Merck Group. It is an imaging flow cytometer that combines the power of flow cytometry with high-resolution microscopy to capture images of individual cells.
Automatically generated - may contain errors
Lab products found in correlation
Ideas software, by Merck Group (2 mentions)
Inspire software, by Merck Group (2 mentions)
Formaldehyde, by Thermo Fisher Scientific (2 mentions)
Draq5, by BioLegend (2 mentions)
Draq5 fluorescent probe, by Thermo Fisher Scientific (2 mentions)
Foxp3 staining buffer kit, by BD (2 mentions)
Accumax, by Merck Group (2 mentions)
Facsdiva 6, by BD (2 mentions)
Draq5, by Cell Signaling Technology (1 mentions)
Cd66b, by BioLegend (1 mentions)
Hetasep solution, by STEMCELL (1 mentions)
Mira 2 lmu, by TESCAN (1 mentions)
Inspire, by Merck Group (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by BioLegend (1 mentions)
Triton x, by Merck Group (1 mentions)
Anti hla dr apc clone l243, by BD (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Retic counttm, by BD (1 mentions)
Draq5, by Biostatus (1 mentions)
Anti cd32 pe, by BioLegend (1 mentions)
26 protocols using imagestream x mark 2 imaging flow cytometer
1
Isolation and Characterization of Neutrophils
2
Bone Marrow Cell Immunophenotyping Workflow
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Sample preparation was performed as previously described42 (link). Briefly, 10 × 106 unfractionated bone marrow cells were fixed using formaldehyde (4%; Alfa Aesar) for 15 min at room temperature. Following two washes with PBS, the cell pellet was cooled on ice for 15 min and permeabilized using ice-cold acetone (a cycle of 50%–100%–50%). Following a wash with PBS (+2% FCS), cells were stained for surface markers. Finally, 10 × 106 cells were resuspended in 100 μL PBS supplemented with DRAQ5 (2.5 μM; BioLegend), with acquisition performed on an ImageStream®X Mark II Imaging Flow Cytometer (Merck) using a ×40 objective lens. Approximately 50,000 events per sample were collected, and data analysis was performed using the associated Image Data Exploration and Analysis software (IDEAS; v.6.2; Merck).
3
Characterization of Magnetite Nanoparticles and Polyelectrolyte Microcapsules
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The morphology and size of magnetite nanoparticles and polyelectrolyte microcapsules were studied by transmission (TEM) and scanning (SEM) electron microscopy. TEM imaging was performed with a FEGTEM microscope (JEOL, Akishima, Tokyo, Japan) operating at 200 kV. Samples for TEM were prepared by drying a drop of the aqueous suspension of nanoparticles on the lacey-carbon copper grid. SEM was performed with MIRA II LMU (Tescan, Brno, Czech Republic) microscope at an operating voltage of 30 kV in secondary and backscattering electron modes. Brightfield and fluorescence images of microcapsules and cells were made using ImageStream X Mark II Imaging Flow Cytometer (Merck, Kenilworth, NJ, USA).
4
Imaging Flow Cytometry Analysis of Stimulated Immune Cells
5
Multiparametric Imaging Flow Cytometry
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Imaging flow cytometry analysis of murine blood samples was performed at the Image Stream Core Facility (Tübingen, Germany) with an Imagestream® X Mark II Imaging Flow Cytometer (Merck KGaA, Darmstadt, Germany). In this approach, fluorescent antibody surface staining is combined with intracellular phalloidin staining of actin following cell permeabilization with 0.1% Triton X (both obtained from Merck KGaA, Darmstadt, Germany).
Antibodies used are listed in the supplemental methods (Supplemental Table 1).
Antibodies used are listed in the supplemental methods (Supplemental Table 1).
6
Flow Cytometric Analysis of B Cell Internalization
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Cells were stained with anti-HLA-DR APC (clone L243; BD Bioscience) in PBS supplemented with 0.1% BSA for 30 min in the dark on ice and fixed for 20 min in PBS with 4% PFA. Primary human B cells or Ramos B cells were washed and 4′,6′-diamidino-2-phenylindole (DAPI; Sigma) was added to stain the cell nucleus. Large particle internalization by human B cells was evaluated on an ImageStreamX mark II imaging flow cytometer (Merck). The acquired data was analyzed using IDEAS V6.2 Software (Merck) and FlowJo Software version 10 (Supplementary Figures 1A , 4 ).
7
Imaging Flow Cytometry Quantification of Lu/BCAM Surface Expression
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Expression of Lu/BCAM on the RBC surface was analyzed using F241 mouse monoclonal antibody. After 1-hour incubation with F241 (dilution [d]: 1/10), the secondary anti-mouse APC-conjugated antibody (d: 1/100) (Beckman Coulter) was added for 1 hour, then RBC were washed and suspended in 200 μL of thiazole orange (TO) dye (Retic-CountTM, Becton-Dickinson) for 30 minutes (min) to label reticulocytes. RBC were analyzed using ImageStream®X Mark II Imaging Flow Cytometer (Merck Millipore) (60x magnification) and the IDEAS software (version 6.2). Lu/BCAM-positive mature RBC (Lu APC) were gated, excluding the reticulocytes (TO-positive events). Using the Modulation Feature, that measures the intensity range of an image, normalized between 0 and 1 (formula: Modulation = Max Pixel - Min Pixel / Max Pixel + Min Pixel), reflecting the fluorescent signal distribution, we defined two subpopulations of mature Lu/BCAM RBC: Low-Modulation (Spots) and High-Modulation (Patches). Based on the x-axis Modulation_M11_Ch11 APC and y-axis Mean Pixel_M11_Ch11 APC, the “Spots” population was between -0.039 and 0.231, and the “Patches” population was between 0.235 and 0.552.
8
Multicolor Flow Cytometry Fusion Assay
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The DIO-labeled HGC-27 cells/SGC-7901 cells and DID-labeled hucMSCs was fused by PEG1500 in vitro and suspended in 200 μl PBS. Then the cell suspensions were analyzed on the Image Stream X Mark IIimaging flow cytometer (Merck Millipore) with low flow rate/high sensitivity. The cell suspensions were acquired immediately and single cell populations were gated for detect the fused cells and unfused cells visually. Four fluorescence channels were visualized in the INSPIRE software: Brightfield images were collected in CH1, DIO fluorescence was recorded using excitation with a 488 nm laser (CH2), and DID fluorescence using excitation with a 640 laser (CH11). A total of 3000–5000 cell events were collected for each sample. Single stained controls were also collected (DIO only and DID only labelled cells) at the same settings in order to develop a compensation matrix for removing spectral overlap of dyes from each of the channels.
9
Quantifying T Cell Nuclear Proteins
10
Immunofluorescence Analysis of RAD51AP1 in SKOV3 and A549 Cells
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A confluent 75 cm2 flask of SKOV3 and A549 cells, respectively, was split evenly into eppendorf tubes, which were centrifuged at 1500 rpm for 5 min. The supernatant was discarded and the pellet was washed with sterile PBS, followed by another spin for 3 min at 2000 rpm. Cells were fixed in 4% paraformaldehyde for 7 min on ice and then centrifuged for 5 min at 1500 rpm. The pellet was washed with PBS and spun at 2000 rpm for 3 min. Cells were blocked for 30 min in FBS-PBS and then centrifuged at 2000 rpm for 3 min. The pellet was incubated overnight with primary antibody (RAD51AP1) diluted in FBS-PBS (1:200) at 4oC. After incubation, cells were centrifuged at 2000 rpm for 3 min and then washed with PBS. Secondary horseradish peroxidase (HRP)-conjugated anti-rabbit antibody diluted in FBS-PBS (1:200) was added to the cells for a 30 min incubation. The cells were centrifuged and washed once more as described above. Accumax (Sigma) and the nuclear marker DRAQ5 (BioStatus) were added before visualizing in the ImageStream. Compensation samples, that only contained antibodies or only DRAQ5, were used for data normalisation. Data and images were analysed using an ImageStreamx Mark II Imaging Flow Cytometer (Amnis, Merck Millipore).
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