Immunocytochemistry of Sorted Cells

The sorted ILC2 and T-cells were fixed in PBS containing 1% paraformaldehyde, washed with PBS, stored, and protected from light until analysis. Sub-membrane actin and nuclei (DNA) were labeled with Alexa Fluor 488 Phalloidin (Sigma-Aldrich, Munich, Germany) and Hoechst 33258 (Sigma-Aldrich), respectively (32 (link)). The cells were either transferred to optical-bottom 12-well plates (Thermo Fisher Scientific) in PBS for observation by confocal microscopy (32 (link)). The samples were analyzed using a Leica DM6000 inverted microscope connected to a Leica SP5 laser scanning confocal system and Fiji software to determine the cell integrity after sorting.

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Palomares F., Pérez-Sánchez N., Nieto N., Núñez R., Cañas J.A., Martín-Astorga M.D., Cruz-Amaya A., Torres M.J., Eguíluz-Gracia I., Mayorga C, & Gómez F. (2024). Group 2 innate lymphoid cells are key in lipid transfer protein allergy pathogenesis. Frontiers in Immunology, 15, 1385101.

Publication 2024

Alexa Fluor 488 Phalloidin Hoechst 33258 DM6000 inverted microscope SP5 laser scanning confocal system

Corresponding Organization :

Other organizations : Andalusian Centre for Nanomedicine and Biotechnology, Instituto de Investigación Biomédica de Málaga, Hospital Regional Universitario de Málaga, Universidad de Málaga

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Variable analysis

independent variables

None explicitly mentioned

dependent variables

Cell integrity after sorting

control variables

Sorting of ILC2 and T-cells
Fixation of cells in PBS containing 1% paraformaldehyde
Washing of cells with PBS
Labeling of sub-membrane actin with Alexa Fluor 488 Phalloidin
Labeling of nuclei (DNA) with Hoechst 33258
Transferring of cells to optical-bottom 12-well plates in PBS for confocal microscopy observation

controls

Positive control: Not specified
Negative control: Not specified

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