Snap cell 647 sir ligand

For the preparation of retrovirus, HEK293T cells were transfected with a retroviral vector together with pCG-VSV-G and pCG-gag-pol (gifts from Dr T. Yasui), using Lipofectamine 2000 (11668019, Thermo Fisher Scientific). Two days after transfection, the supernatant was passed through a 0.45 μm syringe filter unit (SLHV033RB, Merck Millipore, Billerica, MA) and collected. Then, the retrovirus was applied to cells, and stable transformants were selected by application of 2 μg/ml puromycin (P8833, Sigma-Aldrich), 5 μg/ml blasticidin (022-18713, Wako) and 100 μg/ml hygromycin B (10687-010, Thermo Fisher Scientific). For the generation of ATG2A/B DKO HEK293T cells stably expressing ATG2A or its variants, ATG2A/B DKO HEK293T cells were infected with the retrovirus carrying ATG2A or ATG2A variants and the internal ribosomal entry site drive SNAP-tag. Stable transformants were labeled with 100 nm SNAP-Cell 647-SiR ligand (New England Biolabs, S9102S). The fluorescence signals from SNAP-Cell 647-SiR were detected, and cells with the same fluorescence intensities were collected by a cell sorter (MoFlo AstriosEQ; Beckman Coulter).

HaloTag- and SNAP-tag-fused constructs were labeled through incubation with their requisite fluorescent ligand (200 nM JaneliaFluor 646 HaloTag ligand (Promega), or 500 nM SNAP-Cell 647-SiR ligand (New England BioLabs), respectively) in supplemented RPMI-1640 for 30 min at 37°C, washed three times, incubated for a further 30 min then washed once and used immediately for imaging.