Alexa fluor 647 a31573

Cells were cultured on coverslips to 70% confluency and then transfected with plasmids for 48 h. After treatment, cells were prepared for immunostaining by incubation with primary antibodies and then incubated with secondary antibodies conjugated with Alexa Fluor 405 (A31553, 1:300), Alexa Fluor 488 (A11001, 1:300), or Alexa Fluor 647 (A31573, 1:300) (Molecular Probes, OR, USA) for 1 h at 37 °C. F-actin was stained with phalloidin (A12379, Invitrogen) at a 1:40 dilution. Mitochondria and lysosomes were stained with MitoTracker Red CMXRos (M7512, Molecular Probes) and LysoTracker Red DND 99 (L7528, Molecular Probes), respectively, according to the manufacturer’s instructions. Cells were viewed using a laser-scanning confocal microscope (Zeiss, Germany). All images were analyzed by ImageJ software (MD, USA).

2 × 10⁴ THP-1 cells were cultured in supplemented RPMI medium in 96-well plates for 24 h. Then, 8 mg/mL polybrene was added prior to cell transduction with lentiviral particles at a multiplicity of infection (MOI) of 1:10. Cells were incubated for 5 h, and then the culture medium was replaced with fresh medium. After 48 h culture stabilization, fresh medium containing 1.5 mg/mL puromycin was added for 72 h to select transduced cells. The puromycin concentration was then increased to 3 mg/mL, and the cells were incubated for an additional 72 h. AIRE expression in the transduced clones was monitored by western blotting, flow cytometry, and RT-PCR, using anti-AIRE (H-300 and C-2), donkey anti-rabbit Alexa Fluor 647 (a31573) (Molecular Probes), and human AIRE primers (sc-37669-PR). For overexpression of AIRE, 3 × 10⁶ HEK293T cells were transfected with pLenti-AIRE (NM_000383) Human Tagged ORF, mGFP tagged (RC213497L2, OriGene) using FuGENE 6 Transfection Reagent (Promega) for overexpression of AIRE.