Circadian chemical screen assays with human cell lines and mice locomotor assays

Chemical screen assay

This is a lab standard chemical screen protocol for circadian phase perturbation. Basically, the chemical screen was performed in corning 96-well white-bottom plate with Per2-dLuc and Bmal1-dLuc U2OS cells. We define a circadian phase shifter when it causes more than 6 hours phase change while not affect the amplitude. 

Day 1: 2 × 10⁴ cells in 100 μl DMEM culture medium were plated into each well of 96-well white-bottom plate, then sent plates into a 37°C/5% CO₂ air humidified incubator. 

Day 2: 24 hours after cell plating, pre-mixing chemical library which piloted in 96 wells plate with another 96 well plate with 120 μl explant medium each well. The final concentration of chemicals in each well was 10 µM. Then DMEM culture medium was changed into explant medium containing chemicals. Plates were then sealed with sealing film and put into Tecan luminometer. Data from cells harboring Per2-dLuc or Bmal1-dLuc were collected at 36°C for 5~7 days and analyzed with Biodare2 online software.

Note: For each 96 well plate chemicals (DMSO controls were in column 1 and 12 of the 96 well plate), two 96 well plates of Per2-dLuc and two 96 well plates of Bmal1-dLuc U2OS cells were prepared.

Reagent

Solution

Explant medium: Dissolve 1 bag of DMEM powder with 470 ml ddH₂O, supplement with 10 ml HEPES solution, 10 ml Penicillin-Streptomycin solution, 5 ml Sodium Bicarbonate 7.5% solution, 0.1 mM D-luciferin, 2 µg/ml Gentamicin and 5% B27 supplement, adjust the pH to 7.2-7.4, add ddH₂O to 500 ml, filter at 0.22 µm and finally store at 4 °C. 

Equipment

Tecan M200 PRO

Screen used cell culture plate

Corning® 384 well plates, Sigma, CLS3570-10EA

Corning® 96 well plates, clear bottom, Sigma, CLS3917-100EA

Software

Biodare2 (https://biodare2.ed.ac.uk/)

Murine locomotor assay

1. Male WT C57BL/6J mice at age of 2~5 months old were housed individually and entrained under a 12 h light (~ 50 lux light intensity)/12 h dark cycle at 25°C for at least two weeks in a humidity-controlled facility. Locomotor activity was recorded in 5-min bins.

2. One week after the synchronization of mouse clock to ambient light dark cycle, cordycepin or DMSO was dissolved in saline and I.P. injected (15mg/kg) into mouse 1 h before light off (ZT11) or light on (ZT23), then light/dark cycles was 8 h phase advanced or delayed.

3. Cordycepin or DMSO was injected again 24 h later.

4. After 2 weeks continuously recording, the phase of activity onsets was analyzed and recorded with Clocklab software (Actimetrics) following the procedures described as the manufacture. The re-entrainment curves of different treatment group were pictured with Excel, data was presented as mean ± SD.

Note: WT C57BL/6J mice were maintained in specific-pathogen-free (SPF) environment with food/water ad lib under a 12h:12h light-dark (LD) photoperiod. All experiments with mice were performed following the guidelines of the Institutional Animal Care and Use Committee (IACUC) at National Institute of Biological Sciences (NIBS), Beijing, China.

Mice

C57BL/6J mice (2-5 months old), Jackson Labs, JAX Stock#: 000664.

Equipment

SPF mice facility with controllable light, temperature and humidity

Running Wheel Cages with Motion Sensors and Cables, Actimetrics

Reagent

Cordycepin, Santa Cruz Biotech, sc-203902

Software

ClockLab Data Collection, Actimetrics

ClockLab Analysis 6, Actimetrics

Excel, Microsoft

Category