Amplification of PCR products was quantified using FastStart SYBR Green Master (Roche) according to manufacturers instruction and fluorescence montoried on a DNA Engine Peltier Thermal Cycler (Bio-Rad) equipped with a Chromo4 Real-Time PCR Detection System (Bio-Rad) and melting curve analysis also performed.
With objective of identifying either a Piezo1 or Piezo2 agonist, we co-transfected cells with mPiezo1 and mPiezo2 cDNA at equal amounts. 2 days after, transfection cells were incubated with Calcium5 (Molecular Devices, Sunnyvale, CA) according to manufacturer's instruction and fluorescence was monitored on FLIPR Tetra.
Upon hybridization, microarrays were washed and dried according to the Genisphere's instructions and fluorescence measurements were performed using a ScanArrayLite (Perkin Elmer) laser scanner.
Upon hybridization, microarrays were washed and dried according to the Agilent's instructions and fluorescence measurements were performed using an Agilent Technologies G2565B microarray scanner.
Genotyping was carried out in accordance with the kit manufacturer's instructions and fluorescence was measured with a fluorescence polarization reader (Victor-Wallace from Perkin Elmer Life Sciences, Boston, MA, USA) at 20, 25 and 35 cycles.
Alamar Blue (Invitrogen, Carlsbad, CA, USA) was added according to the manufacturer's instructions and fluorescence signal was read using a SpectraMax M4 plate reader (Molecular Devices) using excitation at 535 nm and emission at 590 nm.
Protein quantitation was measured using the RediPlate Protein Quantitation Kit (Molecular Probes) and calibration curves as per manufacturers instructions and fluorescence signals determined using a Fuji Laser Scanner FLA5000.
To measure the fragmented DNA, the nucleotide ends were labeled using a commercial kit (In Situ Cell Death Detection Kit, Peroxidase diaminobenzidine [POD], 11 684 817 910; Roche Diagnostics, Indianapolis, IN, USA) according to the manufacturer's instructions, and fluorescence was visualized by microscopy as described below.
(b) Fluorescence: The Fluor de Lys Assay was used according to the manufacturer's instructions, and the fluorescence was measured on a Tecan Safire 2 fluorescence plate reader.
At different time points, the adherent cells were stained on chamberslides with fluorescein-conjugated annexin V and propidium iodide using Annexin-V-FLUOS Staining Kit (Roche) as per the manufacturer's instructions and the fluorescence of individual cells was assayed by fluorescence microscopy.
The amount of RNA extracted from tissue specimens was measured using the Quant-iT Ribo Green RNA assay (Invitrogen), adhering to the manufacturer's instructions, and measuring fluorescence for 0.1 seconds at excitations and emissions of 485 nm and 535 nm, respectively, using a Victor3 V 1420 Multilabel Counter (Perkin Elmer, Waltham, MA).
