Live cell imaging buffer

Confocal imaging was performed using LSM880 or LSM800 (Carl Zeiss Microscopy) with a 63×/1.40-NA plan-apochromat differential interference contrast oil immersion objective and 32-channel gallium arsenide phosphide photomultiplier tube area detector; 405-, 488-, 561- and 633-nm laser lines were used in this study.
For live imaging, nonneuronal cells were imaged using live cell imaging buffer (Life Technologies), and cultured neurons were imaged in modified Tyrode buffer (119 mM NaCl, 5 mM KCl, 2 mM CaCl₂, 2 mM MgCl₂, 30 mM glucose, 10 mM Hepes, pH 7.35).
Halo ligand, JF646, was used at a final concentration of 200 nM. Cells were incubated with JF646 for 1 h, rinsed, and then, incubated for 30 min (all in culture medium) before imaging in live cell imaging buffer.
For presentation purposes, brightness and contrasts of images were adjusted using the ImageJ software. Some of the high-magnification fields were enlarged using the Adjust Size function on ImageJ.

In total 12,000–16,000 HEK293T cells/well were plated in 100 µL DMEM glutamax (10% FBS) into 96-well plate (P96-1-N, Cellvis), which was precoated with 3.75 µg Poly-D-lysine (30–70 KDa, Alfa Aesar) for 150 min. After 16–20 h, cells were transfected with 80 ng of either control shRNA for every 1st well of the row or shRNAs for the targets of interest following manufacture′s conditions. Briefly, 4.4 µL of opti-MEM containing 0.4 µL of Lipofectamine 3000 was added to mix of 4 µL opti-MEM, 0.16 µL P3000 and 1.6 µL shRNAs mix (50 ng/µL), and resulting DNA:Lipofectamine mix was incubated at room temperature for 13–15 min. After incubation 9.7 µL of the DNA:Lipofectamine mix was added to the corresponding well of the 96-well dish. After 52–56 h from transfection, the cells were washed with 150 µL of Live Cell Imaging Buffer (ThermoFisher) and 70 µL of 1 µM DPP-2 or 500 nM mitoDPP-2 in Live Cell Imaging Buffer was added to the each of the eight wells per row using a multi-channel pipette. The cells were then incubated at room temperature for 10 min and total fluorescence intensity (λ_ex 490/20 nm, λ_em 545/20 nm, Gain 130, read from bottom with height 4.5 mm, and sweep method) was measured at room temperature by scanning the area of each well with a matrix size 11 × 11. The data were normalized to the average fluorescence intensity of the control shRNA.