Digital sight camera ds qi2

To evaluate caspase ‐ induced cell death, the CellEvent Caspase‐3/7 Green Detection Reagent® (Invitrogen #C10423) was used. Briefly, cells were seeded at a concentration of 5,000 cells per square centimetre in 48 wells, using a complete RPMI medium with 10% FBS, 1% of glutamine and 1% of pen/strep antibiotics. Next day, cells were treated with 30 μg/ml of D1 and immediately added with the ready to use fluorogenic substrate to a final concentration of 2 μM, without a washing step. After an incubation of 30 min at 37°C and 5% CO₂, protected from light, cells were imaged under the microscope for 48 hours inside an OKOlab on‐stage incubator in a controlled environment of 37°C, constant humidity and 5% of CO₂, protected from light.
Cells were visualized with a Nikon Inverted Microscope Eclipse Ti‐E, equipped with a Digital Sight camera DS‐Qi2 (Nikon Instruments, Tokyo, Japan) and images were acquired with NIS‐Elements (Nikon Instruments, Tokyo, Japan) software. The whole well was imaged at each time point with automatic stitching performed by the software. Four experiments were performed for each data set and caspase ‐ activated positive cells were counted on six fields of the same size.

To evaluate mitochondrial membrane potential, JC1 was used (Invitrogen #T3168). Briefly, cells were first stained for 30 min at 37°C and 5% CO₂, protected from light, using JC1 at a concentration of 5 μM in RPMI medium. Cells were then seeded at a concentration of 5,000 cells per square centimetre in 48 wells, using complete RPMI medium and treated with 30 μg/ml of D1 extract.
Cells were immediately imaged under the microscope for 24 hr inside an OKOlab on‐stage incubator in a controlled environment of 37°C, constant humidity and 5% of CO₂, protected from light. Cells were visualized with Nikon Inverted Microscope Eclipse Ti‐E equipped with a Digital Sight camera DS‐Qi2 (Nikon Instruments, Tokyo, Japan) and images were acquired with NIS‐Elements (Nikon Instruments, Tokyo, Japan) software. Six experiments were performed for each data set and JC1 ratiometric evaluation was done comparing the evolution of fluorescence signal in the two channels of emission (polarized vs. depolarized mitochondria) over time.