To prepare XB2 keratinocytes for flow cytometry, cells were washed with PBS and fluorescence‐activated cell sorter (FACS) buffer (1% FBS [v/v] and 2 mM EDTA in PBS), centrifuged at 300g for 5 min and resuspended in 200 μl FACS buffer. Cells were incubated with anti‐FLAG‐Cy3 (Sigma) antibody, diluted 1:50 in FACS buffer for 1 h, at 4°C to ensure that internalization was blocked. Cells were then washed with FACS buffer, centrifuged at 300g for 5 min and resuspended with 200 μl FACS buffer. Data acquisition was performed in a FACS CANTO II flow cytometer (BDBiosiences) At least 30 000 cells were acquired per condition using BD FACSDivaTM software (Version 6.1.3, BD Biosciences). Data analysis was performed in FlowJo (Version 10, BD Biosciences).
Anti flag cy3
Manufactured by Merck Group
The Anti-FLAG-Cy3 is a fluorescent-labeled antibody that binds to the FLAG epitope tag. It is commonly used in various research applications for detecting and visualizing proteins tagged with the FLAG sequence.
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Lab products found in correlation
3 protocols using anti flag cy3
1
Flow Cytometric Analysis of FLAG-Tagged Keratinocytes
2
Immunofluorescent Staining of XB2 Keratinocytes
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To prepare XB2 keratinocytes for flow cytometry, cells were washed with PBS and FACS buffer [1% FBS (v/v) and 2 mM EDTA in PBS], centrifuged at 300 x g for 5 minutes and resuspended in 200 μl FACS buffer. Cells were incubated with anti-FLAG-Cy3 (Sigma) antibody, diluted 1:50 in FACS buffer for 1 h, at 4ºC to ensure that internalization was blocked.
Cells were then washed with FACS buffer, centrifuged at 300 x g for 5 minutes and resuspended with 200 μl FACS buffer. Data acquisition was performed in a FACS CANTO II flow cytometer (BDBiosiences) At least 30,000 cells were acquired per condition using BD FACSDivaTM software (Version 6.1.3, BD Biosciences). Data analysis was performed in FlowJo (Version 10, BD Biosciences).
Cells were then washed with FACS buffer, centrifuged at 300 x g for 5 minutes and resuspended with 200 μl FACS buffer. Data acquisition was performed in a FACS CANTO II flow cytometer (BDBiosiences) At least 30,000 cells were acquired per condition using BD FACSDivaTM software (Version 6.1.3, BD Biosciences). Data analysis was performed in FlowJo (Version 10, BD Biosciences).
3
Visualizing WDFY4 and MDA5 in HEK-293 cells
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Immunofluorescence staining HEK-293 cells were grown on coverslips and transfected with the vectors for WDFY4 isoforms and MDA5. Cells were fixed in 4% paraformaldehyde, permeabilized in 0.25% Triton X-100, and stained with anti-V5 and anti-mouse IgG-Alexa488 (Invitrogen) for WDFY4 and anti-FLAG-Cy3 (Sigma-Aldrich) for MDA5 detection. DAPI was used for counter-staining. Images were acquired with the confocal microscope N-SIM (Nikon) using 100X objective lens. The data were analyzed in 3D-SIM mode by using NIS-Elements software (Nikon).
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