The FUHEN cells were washed two times with 1 % HS-αMEM and were suspended with 10 %HS-αMEM, 0.02 % EDTA/PBS, PBS or 0.1 mg/mL MgCl2·6H2O and 0.1 mg/mL CaCl2·2H2O/PBS, 10 % HS-PBS or 10 % HS-EDTA/PBS. Then, the cells were seeded in four petri dishes. After 5 min of incubation at room temperature, the dishes were washed two times with PBS and fixed. Then, the cells were stained with hematoxylin or May-Grünwald Giemsa, and photographs were taken. The number of attached cells per area was counted using ImageJ software. In another experiment, the cells were fixed with 10 % formaldehyde in PBS for 12 h at 4 °C. Then, the cells were treated with 0.1 % Triton X-100 for 5 min at room temperature. The cells were washed with PBS and stained with the actin stain 555 Fluorescent Phalloidin (Cytoskeleton, Inc., Denver, USA) and DAPI, according to the manufacturer’s instructions.
Fluorescent phalloidin
Manufactured by Cytoskeleton
Sourced in United States
Fluorescent phalloidin is a high-affinity probe for filamentous actin (F-actin). It binds tightly to F-actin and can be used to visualize the distribution of actin filaments in cells and tissues.
Automatically generated - may contain errors
3 protocols using fluorescent phalloidin
1
Cell Attachment and Cytoskeleton Visualization
2
Fluorescence and TIRF Microscopy of Cells
3
Fluorescence and TIRF Microscopy of Cellular Fibronectin and Actin
Check if the same lab product or an alternative is used in the 5 most similar protocols
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For fluorescence and total internal reflection fluorescence (TIRF) microscopy, cells were plated onto glass bottom chamber slides, cultured for 2 days in complete media, then serum‐starved for 24 h. Cells were then fixed and stained with antifibronectin antibody (clone FN‐15, Sigma). Actin cytoskeleton was stained with fluorescent phalloidin (Cytoskeleton, Denver, CO, USA) in permeabilized cells. DsRed‐paxillin was imaged in fixed cells or, for TIRF time lapse, in living, serum‐starved cells. Nuclei of fixed cells were counterstained with 1 μg/ml DAPI (Sigma). All fluorescence microscopy was conducted with a Nikon Ti series inverted microscope equipped with a 40× or 60× objective (regular fluorescence) or 60× APO TIRF objective.
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