This study investigates the effects of hydrogel stiffness on the morphology and actin filament formation in adherent neural crest cell cultures. By utilizing fluorescence microscopy, the research highlights the differences in cellular behavior on varying stiffness substrates.
Take adherent neural crest cell cultures grown on coverslips coated with hydrogels of varying stiffness. Wash the cells with buffer.
Add paraformaldehyde to fix the cells and preserve cellular morphology.
Wash the cells with buffer to remove any excess paraformaldehyde.
Next, add a non-ionic detergent to permeabilize the cell membranes.
Wash the cells with buffer to remove excess detergent.
Then, introduce a blocking solution to prevent non-specific binding.
Incubate the cells with a high-affinity filamentous actin probe that binds to actin filaments.
Wash the cells with buffer to remove any unbound probes.
Add a fluorescent nuclear dye to stain the nuclei, then wash with buffer to remove excess dye.
Apply mounting media to the coverslips and examine the stained cells under a fluorescence microscope.
Cells grown on stiffer hydrogels show increased actin filament formation and a more spread-out morphology than those grown on softer hydrogels.
Use tweezers to transport the coverslips to a new plate to minimize false signals from the cells grown directly onto the plate. Then, fix the cells using 500 microliters of 4% paraformaldehyde for 10 minutes, before treating the cells with 500 microliters of 0.1% Triton X-100 at room temperature.
After 15 minutes, block the cells with 250 microliters of 10% Donkey serum. Stain the cells for F-actin with phalloidin at a dilution of 1 to 400 in 250 microliters of 10% Donkey serum, followed by incubation with DAPI for 10 minutes.
Wash the cells with PBS for two minutes before adding three to four drops of mounting medium to each well. On a fluorescence microscope, capture the images of at least three random frames per hydrogel sample producing individual and merged channels.
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