Notes & Tips Multichannel perfusion culture bioluminescence reporter system for long-term detection in living cells Toshiyuki Watanabe a,b , Toshiteru Enomoto a , Masayuki Takahashi a , Sato Honma c , Ken-ichi Honma c , Yoshihiro Ohmiya b,d, * a ATTO, Bunkyo-ku, Tokyo 113-0034, Japan b Department of Photobiology, Hokkaido University Graduate School of Medicine, Kita-Ku, Sapporo 060-8638, Japan c Department of Physiology, Hokkaido University Graduate School of Medicine, Kita-Ku, Sapporo 060-8638, Japan d Research Institute of Genome-Based Biofactory, National Institute of Advanced Industrial Science and Technology (AIST), Toyohiraku, Sapporo 062-8517, Japan article info Article history: Received 1 February 2010 Received in revised form 2 March 2010 Accepted 10 March 2010 Available online 15 March 2010 Keywords: Secreted bioluminescence reporter Perfusion culture Cypridina luciferase Circadian clock abstract We constructed a multichannel perfusion culture system. Using this device, we developed a perfusion device to detect six samples at the same time and demonstrated the response to dexamethasone of the circadian-related promoter activity in Rat1 fibroblast cells. We could detect the sequential phase shifts of the circadian peaks that are dependent on the timing of the drug treatments. We also demon- strate a temporal dual reporter assay using two kinds of secreted luciferase in the perfusion culture. The combination of secreted luciferase and multiple perfusion culture assay system reveals the effects of transient drug treatment for the pharmacological assay. Ó 2010 Elsevier Inc. All rights reserved. Bioluminescence has been used as a reporter to detect promoter activity in prokaryotic and eukaryotic cells. Recently, biolumines- cence reporter assays have been applied to long-term real-time monitoring of promoter activity in living cells. In particular, firefly luciferase is a useful reporter for long-term monitoring because firefly luciferin retains its substrate activity for a long time in cul- ture [1–3]. However, because the firefly bioluminescence reaction consumes luciferin, adenosine-5 0 -triphosphate (ATP), 1 and oxygen and is affected by fluctuations in pH, it is unsuitable for evaluating the response to any drugs that may affect the intercellular condition via the extracellular environment [4–6]. To avoid this problem, the use of secreted luciferases as reporter enzymes is focused on ex vivo reporter assays. Secreted luciferases have been cloned from luminous organisms [7–11]. After the transfection, these luciferases can be secreted by mammalian cells. Secreted luciferase is a good tool for monitoring circadian rhythm ex vivo [12]. We previously established a perfusion culture system to continuously detect clock gene Bmal1 promoter activity using a secreted Cypridina luciferase (CLuc) expressed in Rat1 fibro- blast cells and succeeded in monitoring circadian fluctuations in the activity of its promoter in the cell culture medium [13]. However, the practicality and reproducibility of the system were limited be- cause of the difficulty in making this system multiple channels. To solve these problems, we constructed a multichannel perfu- sion culture system corresponding to six flow paths (Fig. 1A; see also Fig. S1 in Supplementary material). This system can collect six perfusion samples at the same time with two multiperistaltic pumps that correspond to six flow paths, valves, an incubator, and fraction collectors. One peristaltic pump was used to insert the culture medium from bottles into the cell culture plate, and the other one was used to collect medium from the cell culture plates and distribute this to the fraction collectors. We could select whether to operate these pumps continuously or intermittently, and the flow rate of the perfusion was kept at 0.1–8.0 ml/h. We also made a perfusion chamber with a 12-well cell culture plate. To keep each well from drying up, the tips of the output port were adjusted to 3 mm above the bottom of the culture plate. Two pumps enable us to choose how to perfuse medium—either contin- uously or intermittently (Fig. 1B and C). For the continuous perfu- sion culture, two peristaltic pumps move constantly and simultaneously. For the intermittent perfusion culture, after col- lecting culture medium by pump B, pump A starts to add fresh medium to the cell culture plate. To transiently insert the medium containing the reagent, we used branching silicon tubes that led to 0003-2697/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2010.03.014 * Corresponding author at: Department of Photobiology, Hokkaido University Graduate School of Medicine, Kita-Ku, Sapporo 060-8638, Japan. Fax: +81 72 751 8370. E-mail address: y-ohmiya@aist.go.jp (Y. Ohmiya). 1 Abbreviations used: ATP, adenosine-5 0 -triphosphate; CLuc, Cypridina luciferase; DMEM, Dulbecco’s modified Eagle’s medium; FBS, fetal bovine serum; Dex, dexa- methasone; GLuc, Gaussia luciferase; CMV, cytomegalovirus. Analytical Biochemistry 402 (2010) 107–109 Contents lists available at ScienceDirect Analytical Biochemistry journal homepage: www.elsevier.com/locate/yabio