Rg221408

Human astrocytes were obtained from N7805100 (Thermo Fisher Scientific (Waltham, MA, USA)) and #1800 (ScienCell Research Laboratories (Carlsbad, CA, USA)). Human astrocytes are normal human cells derived from human brain tissue. Human astrocytes were cultured in Gibco™ Astrocyte Medium containing N-2 Supplement, Dulbecco’s modified Eagle medium (DMEM), 10% (vol/vol) One Shot™ Fetal Bovine Serum (FBS), 100 units/mL penicillin and 100 mg/mL streptomycin (A1261301, Thermo Fisher Scientific (Waltham, MA, USA)). For overexpression of human CLOCK or BMAL1, human astrocytes were seeded and transduced with pCMV6-AC-GFP constructs against human CLOCK (NM_004898) (RG221408, Origene (Rockville, MD, USA)), pCMV6-AC-GFP constructs against human BMAL1 (ARNTL) (NM_001178) (RG207870, Origene (Rockville, MD, USA)) or pCMV6-AC-GFP vector (PS100010, Origene).

Cell cytotoxicity was measured from culture medium of human astrocytes by LDH-Cytotoxicity Colorimetric Assay Kit II (#K313-500, BioVision (Milpitas, CA, USA)) following the manufacturer’s instructions. Human astrocytes (2 × 10⁵ cells in 6-well cell culture plate) were seeded and transduced with pCMV6-AC-GFP constructs against human CLOCK (NM_004898) (RG221408, Origene (Rockville, MD, USA)), pCMV6-AC-GFP constructs against human BMAL1 (ARNTL) (NM_001178) (RG207870, Origene (Rockville, MD, USA)) or pCMV6-AC-GFP vector (PS100010, Origene). Cells were incubated for 24 h.

Human astrocytes (2 × 10⁴ cells) were seeded in FluoroDishTM (FD35-100, World Precision Instruments (Sarasota, FL, USA)). Cell were transduced with pCMV6-AC-GFP constructs against human CLOCK (NM_004898) (RG221408, Origene (Rockville, MD, USA)), pCMV6-AC-GFP constructs against human BMAL1 (ARNTL) (NM_001178) (RG207870, Origene (Rockville, MD, USA)) or pCMV6-AC-GFP vector (PS100010, Origene). Cells were incubated for 24 h or 48 h. Three-dimensional images were analyzed by 3D Cell Explorer (NANOLIVE (Ecublens, Switzerland)). The images were representative images from a total of 100 cells in ten individual images per group.