HaCaT cells were seeded 24 hours before transfection at a cell density of 5 × 104 cells per well on coverslips in 12-well plates. Twelve hours after transfection, cells were switched to DMEM without calcium supplemented with 1% FBS and then 12 hours after DMEM with 0.1% FBS. This was followed by treatment with dimethyl sulfoxide (DMSO; vehicle), EGF (100 ng/ml) for 30 s (to induce formation of filopodia), EGF (10 ng/ml) for 2 min (to induce formation of lamellipodia), or CN01 (1 U/ml; Cytoskeleton Inc.) for 5 min (to induce formation of stress fibers). All cells were then washed in PBS, fixed in 4% paraformaldehyde for 10 min, and permeabilized using 0.5% Triton X-100/PBS for 5 min. To visualize actin cytoskeleton, cells were incubated with 100 nM rhodamine phalloidin (Cytoskeleton Inc.) and imaged using an epifluorescent Nikon Eclipse E400 microscope equipped with a QImaging QICAM 12-bit Fast 1394 camera (Nikon).
Epidermal growth factor (egf)
Manufactured by Cytoskeleton
EGF (Epidermal Growth Factor) is a recombinant protein product from Cytoskeleton. It is a polypeptide that stimulates cell growth and differentiation.
Automatically generated - may contain errors
Lab products found in correlation
2 protocols using epidermal growth factor (egf)
1
Visualizing Actin Cytoskeleton Dynamics
2
Pharmacological Manipulation and Calcium Chelation in Retinal Waves
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For pharmacology experiments, after 5–10 min of recording data in ACSF, pharmacological agents were added to the perfusion, and experimental recordings were obtained 5 min afterward. Drug concentrations were as follows: 5 µM cytochalasin-D (Avantor), 10 µM nocodazole (Sigma-Aldrich), 1 unit (100 ng)/ml EGF (Cytoskeleton, Inc), 5 µM pirenzepine (Tocris), 5 µM gabazine (Tocris), 25 µM DL-TBOA (Tocris), 20 µM DNQX (Tocris), 50 µM AP5 (Tocris), and 200 µM BAPTA-AM (Tocris). DL-TBOA and BAPTA-AM were prepared in 0.1% DMSO. For calcium chelation experiments, whole-mount retinas were incubated in BAPTA-AM or vehicle (ACSF/0.1% DMSO) for 1.5–2 hr, and then moved to ACSF for 30 min prior to imaging (Shigetomi et al., 2008 (link)). To verify that BAPTA-AM loading abolished retinal waves and residual calcium activity in cytosolic and membrane-proximal compartments, we measured calcium activity in neurons and Müller glia using cyto-GCaMP6fflox (crossed with PDGFRα-Cre) or Lck-GCaMP6fflox (crossed with Slc1a3-CreER) at P10–P12 before and after BAPTA-AM loading.
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