AAV vector preparation
AAV9 vector carrying GCaMP under the control of the Tet-Off system was produced in HEK293 cells using the calcium phosphate transfection method. Cells cultured in seven 15-cm dishes were harvested to purify AAV particles using the AAVpro Purification Kit Maxi (All Serotypes) (Cat. # 6666). Dulbecco's phosphate-buffered saline was used in place of the kit's Suspension Buffer, and the AAV particles were concentrated to ~125 µl using an Amicon Ultra-4 Centrifugal Filter Unit* (Amicon, Cat. # UFC810008). Viral titer was measured by qPCR (~1 x 1014 GC/ml).
*The Amicon Ultra-4 Centrifugal Filter Unit, which has a typical final concentrate volume of 50 µl, is not a component of the AAVpro Purification Kit Maxi (All Serotypes) (Cat. # 6666). The kit includes the Amicon Ultra-15, 100 kDa Centrifugal Filter Unit, with a typical final concentrate volume of 200 µl.
Injection of the prepared AAV vector into mouse visual cortex
~800 nl of the purified AAV vector was injected into mouse visual cortex. Approximately one week after injection, GCaMP expression was observed in vivo using light microscopy oriented above a glass window covering the injected region. Fluorescence at the cellular level was observed at a 200–300 µm depth in the same region of the visual cortex using a two-photon microscope.