Surface-stained mouse splenocytes (1 x 106) were fixed in 4% paraformaldehyde. An Amnis ImageStream Mark II (EMD Millipore, ON, Canada) was used to collect at least 10,000 images for each sample as we have previously described (31 (link)). Data were analyzed using the IDEAS analysis software package to select single-cell images that were in focus. Next, we performed gating for NK cells (CD49b+) followed by gating for cells that co-express Gal-9 and CD44. Colocalization of Gal-9 and CD44 was determined using the IDEAS colocalization wizard.
Amnis imagestream mark 2
Manufactured by Merck Group
Sourced in United States, Canada
The Amnis ImageStream Mark II is a high-speed, multispectral imaging flow cytometer that combines the power of flow cytometry and digital microscopy. It captures multicolor images of individual cells in flow, providing rich data on cellular morphology and intracellular details.
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Lab products found in correlation
Fv1000mpe, by Olympus (1 mentions)
Ebioscience transcription factor staining buffer set, by Thermo Fisher Scientific (1 mentions)
Pe conjugate, by Cell Signaling Technology (1 mentions)
Interleukin 7 (il 7), by Thermo Fisher Scientific (1 mentions)
Mojosort mouse hematopoietic progenitor cell isolation kit, by BioLegend (1 mentions)
Il 23, by R&D Systems (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Ideas software, by Merck Group (1 mentions)
Fasl pe, by Thermo Fisher Scientific (1 mentions)
Esat 6, by BEI Resources (1 mentions)
9 protocols using amnis imagestream mark 2
1
Gal-9 and CD44 Colocalization in NK Cells
2
Co-expression analysis of donor and lung cells
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Confocal microscope (Olympus FV1000MPE, Japan) and imaging flow cytometry (Amnis ImageStream MarkII, Merck Millipore, USA) were utilized to analyze the co-expression between donor marker (tdTomato-red fluorescence) and lung epithelial cell markers. For confocal microscope, frozen section of lung tissue was made after the cardiac perfusion of mice. Lung epithelial cell and endothelial cell markers (including Pan-keratin, T1-α, SP-C, E-Cadherin and CD31) conjugated with FITC and DAPI were stained respectively and observed in laser confocal microscope. The co-localization of PE and recipient lung cells were inspected. For image flow cytometry, lung tissue was digested into single cells and stained with lung epithelial and endothelial cell marker (including T1-α, SP-C and CD31) conjugated with APC, DAPI, and exclusion with blood cells (described as above Mix and CD45). The co-localization of PE fluorescence and lung epithelial/endothelial cells were quantified by the image flow wizard software.
3
IFN-Induced STAT1 Localization in HeLa Cells
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Hela cells transfected with NC or ASOs were stimulated with IFN (1,000 units/ml) for 30 min and then fixed and permeated with eBioscience transcription factor staining buffer set (Invitrogen) according to the manufacturer's protocol. Cells were resuspended in 100 μl of FACS buffer and incubated with antibody (diluted by 1:50) for at least 30 min at room temperature in dark. DAPI was used to stain cell nuclei for <3 min. Cells were then washed and resuspended in FACS buffer (volume between 20 and 200 μl) in an appropriate cell concentration of 1–2 × 107/ml. Cell samples were loaded and analyzed using Amnis ImageStream MarkII (Merck). Similarity between STAT1 and nuclei staining pattern were calculated. The antibody used in flow imaging cytometry experiment was STAT1 rabbit mAb (PE Conjugate; Cell Signaling Technology).
4
Imaging Flow Cytometry Analysis of PBMCs
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PBMCs were surface stained and fixed with PFA for ImageStream analysis. We collected ≥3000 images for each condition using Amnis ImageStream Mark II (EMD Millipore). Analysis was performed by choosing a high aspect ratio, choosing only in focus images and calculating maximum pixel intensity of the fluorochrome dye according to our previous report.25 32 (link)
5
High-throughput Cell Analysis via ImageStream
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An Amnis ImageStream Mark II (EMD Millipore) was used to collect at least 5,000 images for each sample and each condition. Analysis was performed by selecting fluorescence intensity for each targeted cell marker.
6
Visualizing FOXO1 Localization in Intestinal ILCs
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To visualize intracellular localization of FOXO1 protein, ILCs in small intestine from WT mice were isolated using MojoSort Mouse Hematopoietic Progenitor Cell Isolation Kit (480003; BioLegend) and cultured in RPMI1640 complete medium supplemented with 10% FBS, 1× penicillin-streptomycin, 2 mM glutamine, 25 ng ml−1 IL-7 (PeproTech), 25 ng ml−1 IL-23 (R&D), and vehicle or cAMP (50 μM) for 16 h. ILCs were stained with PI, FOXO1, Lin (CD3, CD19, CD11b, CD11c, Gr-1, TER-119, TCRβ, TCRγδ, NK1.1), CD45, and RORγt, and analyzed by imaging flow cytometry (Amnis ImageStream MarkII; Merck). The nuclear localization of FOXO1 was analyzed using the IDEAS software v.6.2 (Merck).
7
Multiparametric Flow Cytometry Analysis
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Stained cells (1 × 106) were fixed in 4% paraformaldehyde, and data were acquired on an Amnis Image Stream Mark II (EMD Millipore, ON, Canada) according to our previous protocols [52 (link),53 ]. A minimum of 10,000 events were acquired for each sample. Data were analyzed using the IDEAS Analysis Software Package. Neutrophils were identified as having an area >100 and positively stained for CD15. T cells were identified by having an area >80 and stained positively for CD3. Colocalization was measured by calculating the Bright Detail Similarity of the colocalizing probes using the IDEAS wizard. Capping was measured using a delta centroid XY calculation.
8
Morphology of Lin- CD123+ Cells
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The morphology of total pre-enriched Lin− CD123+ cells was evaluated in PBMCs previous to depletion of Lineage+ cells (as described above) and stained with anti-CD123 and the nuclear dye DAPI (Thermo Fisher Scientific, MA, USA). Cells were acquired using the Amnis ImageStream Mark II and analyzed by the IDEAS® software (Merck-Millipore, MA, USA).
9
Investigating Antigen-Specific T Cell Responses
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Isolated CD4 þ T cells were activated with ESAT-6 (BEI Resources) and retinal crude extract, respectively, in the absence of CD28, for 10 hours under standard cell culture conditions. Then cells were stained for intra/extra cellular proteins, FasL-PE (Invitrogen), LAMP-1 FITC (eBioscience). Samples were acquired and analyzed by using Amnis Image-Stream Mark II (Merck Millipore, Seattle, WA, USA) with appropriate controls.
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