Detecting Hydrogen Peroxide Levels in Cultured Neurons Through Live-Cell Fluorescence Imaging
Trascrizione
On the day of imaging, check cells under the microscope to validate growth of RGC axons. For live-cell imaging, transfer the coverslips from the culture dish to a live-cell imaging chamber. Use an inverted microscope equipped with a DIC objective, OG590 long-pass red filter, and an EM-CCD camera.
Before imaging, replace the ZFCM+ medium with ZFCM-. After positioning the cells with the 10x objective, acquire images at 60x magnification using an oil immersion objective. Use an additional 1.5x magnification.
First, acquire DIC images, then image roGFP2-Orp1 using an appropriate filter set. After taking the first set of images, exchange media with media containing different treatment solutions. Media should be changed every 30 minutes of imaging to avoid pH and osmolarity changes.
