Computational protocol: Rapid and Localized Mechanical Stimulation and Adhesion Assay: TRPM7 Involvement in Calcium Signaling and Cell Adhesion

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Protocol publication

[…] Cells electroporated with siRNA were lysated after two days of incubation and had their TRPM7 (210 kDa) and Glyceraldehyde 3-Phosphate Dehydrogenase (GAPDH, 37 kDa) expression examined by Western blot to verify the inhibition of TRPM7 expression by siRNA. 16 μg of total proteins in 2% SDS loading buffer were separated by polyacrylamide gel (10% mixture) electrophoresis, along with a pre-stained protein standard (Invitrogen, 10748010), and transferred to a nitrocellulose membrane (Bio-Rad, 162–0115). After blocking unspecific binding with 5% milk for one hour, the membrane was cut and incubated overnight at 4°C on a rocker with primary antibodies: 1:1000 goat anti-TRPM7 (abcam, ab729) for the top part (>60 kDa) and 1:1000 goat anti-GAPDH (Santa Cruz) for the bottom part (<60 kDa). Then, the membrane was incubated in the horseradish peroxidase-conjugated secondary antibody donkey anti-goat immunoglobulin (Santa Cruz, SC-2020) at 1:5000 for one hour at room temperature on a shaker. The protein expression was observed with SuperSignal West Femto enhanced chemiluminescence (ECL) substrate (Thermo Scientific, 34094). Each part of the membrane was photographed with the necessary exposure to allow a clear image but avoid saturation, according to the amount of expression. ImageJ was used for background subtraction and the integration of chemiluminescence from TRPM7, which was normalized by the GAPDH integration. [...] Live cell images of the biosensors were captured and analyzed by MetaFluor 6.3r7. The fluorescence of the calcium biosensor FRET pair was observed in a circular region of interest, with 50 pixels of diameter, as close as possible to the area stimulated by the probe (). The FRET ratio, directly related to [Ca2+]i, was calculated by dividing the average intensity of YPet by that of ECFP. A region not covered by the cells and close to the probe tip was selected to assess the background signal, which was subtracted from the image. The change in [Ca2+]i reported by the biosensor after mechanical stimulation was quantified as a relative difference (RelDiff) to basal measurements (before stimulation): RelDiff=maxRatio−basalRatiobasalRatio, where maxRatio is the maximum Ypet/ECFP ratio after stimulation and basalRatio is the average Ypet/ECFP ratio before stimulation.To characterize the vibration, images from the fast camera were analyzed using an ImageJ (available at http://rsb.info.nih.gov/ij; developed by Wayne Rasband, National Institutes of Health, Bethesda, MD) plugin, bUnwarpJ []. This plugin was used to compare the position of the beads between two frames: a frame displaced by the probe motion and a reference frame with the probe merely positioned but not moving. Matlab (MathWorks) was applied to reconstruct the displacement map obtained from bUnwarpJ, plotting it as a colormap combined with the vectors for a convenient and clear understanding of the data. A colormap for the strain at each point was also plotted, with vectors denoting the displacement. The total strain E was calculated as: E=Ex2+Ey2, where E x and E y are respectively the strain in x and y axis, or Ex=duxdxandEy=duydy, where u x and u y are the displacements in x and y axis, with each derivative numerically calculated by finite differences (2-point estimation for the boundaries, 3-point estimation for interior points). […]

Pipeline specifications

Software tools ImageJ, bUnwarpJ
Applications Microscopic phenotype analysis, FRET
Organisms Homo sapiens
Chemicals Calcium