Cells (1.5 × 105 cells/well) were seeded on a glass coverslip in a 24-multiwell plate. The next day, cells were washed three times with phosphate-buffered saline (PBS), fixed in 4% paraformaldehyde for 10 min at room temperature (RT), and washed twice with PBS. Cells were first permeabilized with PBS + 0.1% X-100 Triton (93418; Merck KGaA) for 10 min, and then incubated in blocking buffer (3% bovine serum albumin, A7906; Merck KGaA) + 0.3 M glycine (G-7126-500-50; Merck KGaA) for 30 min at RT. Subsequently, cells were treated for 1.5 h in the dark at RT with the following labeled antibodies: anti-cluster of differentiation 49a (CD49a) (130-101-397), anti-CD49f (130-097-246), anti-CD184 (130-098-354), anti-epithelial cell adhesion molecule (EpCAM) (130-091-253), and anti-cytokeratin 19 (CK19; 130-080-101). All antibodies were purchased by Miltenyi Biotec B.V. & Co. KG (Bergisch Gladbach, North Rhine-Westphalia, Germany), and labeled with phycoerythrin, except anti-CK19, which was labeled with fluorescein isothiocyanate. Nuclei were counterstained with 4′,6-diamidino-2-phenylindole included in mounting medium (ab104139; Abcam plc, Cambridge, United Kingdom). Cells were analyzed using the Nikon Eclipse Ti-E confocal microscope and by flow cytometry analysis using the FlowSight Cytometer (Amnis; Merck Millipore) and the IDEAS software.
Flowsight cytometer
Manufactured by Merck Group
Sourced in United States, Germany
The FlowSight cytometer is a flow imaging instrument designed for cellular analysis. It captures high-resolution images of individual cells as they flow through the system, providing detailed information about cell morphology and fluorescence. The core function of the FlowSight is to enable the visualization and quantification of cellular characteristics within a sample.
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Lab products found in correlation
3 protocols using flowsight cytometer
1
Phenotypic Characterization of Cultured Cells
2
Quantifying DNA Damage and Cell Cycle
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Phosphorylated H2AX (γH2AX) was measured to assess DNA damage. At 2, 24, and 36 h after carbon-ion irradiation, cells were trypsinized and collected by centrifugation. Subsequently, 1 × 106 cells per sample were treated with the transcription factor staining buffer kit and then treated cells were incubated with the anti-γH2AX antibody (Ser-139) overnight at 4 °C. Samples were then washed and resuspended for 1 h in Alexa-488 conjugated secondary antibody. After a second rinsing, cells were resuspended in PBS and analyzed using a FlowSight cytometer (Amnis, Merck Millipore, Burlington, MA, USA), and the images were analyzed using ImageStream Data Exploration and Analysis Software (IDEAS, Merck Millipore) and the mean fluorescence intensity (MFI) was calculated. For each sample, 20,000 cells were considered.
Ethanol-fixed samples were rehydrated, washed twice with PBS, and treated with the cell cycle kit according to the manufacturer’s instructions. Cell cycle distribution was then analyzed with FlowJo 7.6.1 software (Tree Star, Portland, OR, USA) using the original histogram of DNA content as measured with the FlowSight cytometer. For each sample, 20,000 cells were considered.
Ethanol-fixed samples were rehydrated, washed twice with PBS, and treated with the cell cycle kit according to the manufacturer’s instructions. Cell cycle distribution was then analyzed with FlowJo 7.6.1 software (Tree Star, Portland, OR, USA) using the original histogram of DNA content as measured with the FlowSight cytometer. For each sample, 20,000 cells were considered.
3
Flow Cytometry Analysis of Cellular Digestion
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The cell digestion, fixation, transmembrane and incubation procedures were the same as flow cytometry assays according to the previous report 19 (link). Cells were analyzed by a FlowSight cytometer (Merck Millipore, Germany).
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