Cell Surface-Anchored Fluorescent Aptamer Sensor Enables. Imaging of Chemical Transmitter Dynamics

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1 Supporting Information for Cell Surface-Anchored Fluorescent Aptamer Sensor Enables Imaging of Chemical Transmitter Dynamics Takeshi Tokunaga, Shigeyuki Namiki, Katsuhiro Yamada, Takahiro Imaishi, Hiroshi Nonaka, Kenzo Hirose, and Shinsuke Sando*,, INAMORI Frontier Research Center, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka , Japan, Department of Neurobiology, Graduate School of Medicine, The University of Tokyo, Bunkyo-ku, Tokyo , Japan, and PRESTO, Japan Science and Technology Agency, Honcho, Kawaguchi, Saitama , Japan. CONTENTS: 1. Supporting figures 2. Methods 2-1. General 2-2. Experiments using HeLa cells Cell culture Immobilization of bio-fapt, toc-fapt, and toc-frdmodn Apparent dissociation constant MTT viability assay 2-3. Experiments using rat brain astrocytes Cell culture Immobilization of toc-fapt and toc-frdmodn Apparent dissociation constant Substrate selectivity Fluorescence change upon quick buffer exchange Visualization of Ade and calcium dynamics on astrocytes 2-4. Experiments using mouse brain astrocytes Cell culture Imaging Ade release by hypotonic / thrombin stimulation S1

2 1. Supporting figures Figure S1. Kinetics for the interaction of bio-fapt and ATP. (a) Typical example of timedependent change in fluorescence intensity of bio-fapt (0.2 µm) upon addition of ATP (500 µm) using stopped-flow mixing (SF-61 DX2 Double-mixing Stopped-Flow System, HI-TECH SCIENTIFIC), observed at 25 C in HEPES buffer (ph 7.4) containing 25 mm HEPES, 119 mm NaCl, 5 mm KCl, 2 mm CaCl 2, 2 mm MgCl 2, and 30 mm glucose. (b) Concentration dependence of the observed rate constant (k obs ) for the complexation of bio-fapt and ATP. The data were obtained by mixing 0.025, 0.05, 0.1, 0.15, and 0.2 µm bio-fapt with 63, 125, 250, 375, and 500 µm ATP, respectively. Kinetic rate constants, k on and k off, were determined as the slope and intercept of the least-squares line. Figure S2. Bright-field (BF, left) and confocal fluorescent (FL, right) images of bio-fapt-hela cells. S2

3 Figure S3. (a) Ade-dependent fluorescence enhancement of fapt (ex. 495 nm, em. 518 nm) in solution. Error bars show standard deviations (n = 3). (b) Fluorescence enhancement of fapt (ex. 495 nm, em. 518 nm) in the presence of nucleotide triphosphates (200 µm each). Error bars show standard deviations (n = 3). Experiments were performed in 25 mm HEPES buffer (ph 7.4) containing 119 mm NaCl, 5 mm KCl, 2 mm CaCl 2, 2 mm MgCl 2, and 30 mm glucose. S3

4 Figure S4. Confocal large area images of (top) toc-fapt-hela and (bottom) toc-frdmodnhela (left) before and (right) after addition of 200 µm of ATP. S4

5 Figure S5. MTT viability assay of toc-fapt-hela cells. Error bars show standard deviations (n = 4). Figure S6. Time course of fluorescence enhancement of toc-fapt-ast cells (red line) with or (blue line) without hypotonic stress and thrombin addition. Error bars show standard errors. S5

6 2. Methods 2-1. General Reagents and solvents were purchased from standard suppliers and used without further purification. All DNAs were purchased from Gene Design Inc. (Japan). Confocal fluorescence imaging experiments shown in Figures 4b and S2 and S4 were performed on a confocal laserscanning microscope (LSM510 META, Carl Zeiss) equipped with appropriate filters. Other fluorescence imaging experiments, including quantitative analyses, were performed on an inverted microscope (IX-71; Olympus) equipped with CCD cameras (ixon, Andor) and appropriate filters. Image analysis was performed using ImageJ software (NIH). Buffer conditions were as follows. HBS: HEPES buffer (ph 7.4) containing 25 mm HEPES, 119 mm NaCl, 5 mm KCl, 2 mm CaCl 2, 2 mm MgCl 2, and 30 mm glucose; and HBS!: HEPES buffer (ph 7.4) containing 25 mm HEPES, 119 mm NaCl, 2.5 mm KCl, 2 mm CaCl 2, 1 mm MgCl 2, 30 mm glucose, and 10 mm sodium ascorbate Experiments using HeLa cells Cell Culture HeLa cells were typically cultured in 5% CO 2 in a humidified incubator at 37 C in Dulbecco s modified Eagle s medium (DMEM) supplemented with 5 10% fetal bovine serum and 1% penicillin streptomycin. HeLa cells were trypsinized, diluted with fresh medium, and transferred to 35 mm glass-based dishes (for Figure S2, 4b, S4), glass cover slips coated with poly-l-lysine (for determination of dissociation constants), or 24-well plates coated with poly- L-lysine (for MTT assay) Immobilization of bio-fapt, toc-fapt, and toc-frdmodn S6

7 Preparation of bio-fapt-hela cells: HeLa cells were incubated with 200 µm of NHS-PEG 4 - Biotin (Quanta BioDesign or Thermo Scientific) in HBS for 30 min at room temperature. After the biotinylation, cells were washed with HBS. The complex of bio-fapt (455 nm) and streptavidin (1.72 µm), which was prepared by incubation for 20 min at room temperature in HBS, was added to the biotinylated cells. After 20 min of incubation, the cells were washed with HBS and then used for subsequent experiments. Preparation of toc-fapt- or toc-frdmodn-hela cells: HeLa cells were treated with 455 nm toc-fapt or toc-frdmodn in HBS at 4 C. After 30 min of incubation, the cells were washed with HBS and then used for subsequent experiments Apparent dissociation constant bio-fapt-hela cell culture was prepared as mentioned above. The cell culture solution was replaced with HBS containing an appropriate concentration of ATP by perfusion. Fluorescence images were taken at each concentration of ATP. As discussed in main text, the ATP-sensing with fapt on cell surface was roughly assumed as a pseudo one-to-one model. Dose-dependent changes in fluorescence intensity were fitted using the equation ax/(k d + x), in which a, x, and K d represent the maximal changes in normalized fluorescence, ATP concentration, and the dissociation constant between bio-fapt-hela cells and ATP, respectively. Quantitative data was calculated from the large area image including several tens of cells (n = 6) MTT viability assay One day before toc-fapt immobilization, HeLa cells were seeded onto poly-l-lysine-coated 24-well plates (2 " 10 5 /ml). toc-fapt was immobilized on HeLa cells (toc-fapt-hela) as S7

8 mentioned above. The cells treated by HBS without toc-fapt were set as the control (control- HeLa). MTT viability assays were performed using a Colorimetric (MTT) Kit For Cell Survival and Proliferation (Millipore) according to the manufacturer s protocol. The standard deviation was calculated for four experiments Experiments using rat brain astrocytes Cell Culture Rat astrocytes were obtained from E21 Sprague Dawley rats. The embryos were decapitated, and the brains were dissociated, finely minced, and trypsinized. The cells were seeded onto collagen-coated plastic dishes in DMEM with 10% FBS, 4 mm glutamine, 1 mm pyruvate, and penicillin streptomycin, and used after ~2 weeks. Astrocytes were seeded onto glass cover slips coated with poly-l-lysine and laminin before imaging. All animal experiments were performed according to the Institutional Guidance of The University of Tokyo on Animal Experimentation and under permission by the animal experiment committee of The University of Tokyo Immobilization of toc-fapt and toc-frdmodn Rat astrocytes were treated with 455 nm toc-fapt or toc-frdmodn in HBS at 4 C for 15 min. After incubation, cells were washed with HBS and then used for subsequent experiments Apparent dissociation constant toc-fapt-ast was prepared as mentioned above. Experiments and analyses were performed using the same procedure described in The standard deviation was calculated for four experiments. Quantitative data was calculated from the large area image including several tens S8

9 of cells (n = 4) Substrate selectivity toc-fapt-ast was prepared as mentioned above. The cell culture solution was replaced with HBS containing 200 µm of the appropriate nucleotide by perfusion. Fluorescence images were obtained in the absence or presence of nucleotides. The standard deviation was calculated for four experiments. Quantitative data were calculated from the large area images including several tens of cells (n = 4) Fluorescence change upon quick buffer exchange After immobilization of toc-fapt onto the astrocyte surface, various concentrations of ATP in HBS! were puffed onto cells using a homemade puffing pipette for rapid solution exchange (within ~2 s). Fluorescence images were acquired at an acquisition rate of 2 Hz Visualization of Ade and calcium dynamics on astrocytes Rat astrocytes were loaded with X-Rhod1-AM (2 µm) by incubation for 30 min at room temperature and were then incubated with toc-fapt (0.21 µm) for 15 min at 4 C. Dual-color imaging of Ade and calcium was performed on an inverted microscope (IX-71; Olympus) equipped with two CCD cameras (ixon, Andor) to simultaneously capture the two images. The specimens were illuminated through a "40 objective lens (NA 0.95, Olympus) by two color excitation lights ( and nm) with a dual-band filter set comprising an excitation filter (468/553 nm BrightLine dual-band band-pass filter, Semrock) and a dichroic mirror (493/574 nm BrightLine dual-edge dichroic beam-splitter, Semrock). Fluorescence light captured by the same objective lens was split by a 570 nm dichroic mirror (Olympus) and S9

10 directed into the two CCD cameras through green ( nm) and red ( nm) emission filters (Olympus). Image pairs were acquired at an acquisition rate of 1 Hz. Mechanical stimulation was applied to astrocytes by gently touching them with a glass pipette using micromanipulators in HBS!. Images representing the fractional increase in fluorescence were generated by dividing every fluorescence image after mechanical stimulation by the averaged images before stimulation Experiments using mouse brain astrocytes Cell Culture Mouse astrocytes were obtained from P0 ICR mouse. The pups were decapitated, and then cortex were obtained immediately. Cortex were treated with papain for 10 min at 37 C. Dissociated cells were seeded onto plastic dishes in DMEM with 10% FBS and penicillin streptomycin. After one week culture, cells were seeded on glass cover slips coated with poly- L-lysine for imaging experiments. All animal experiments were performed according to the Institutional Guidance of The University of Tokyo on Animal Experimentation and under permission by the animal experiment committee of The University of Tokyo Imaging Ade release by hypotonic / thrombin stimulation Mouse astrocytes were removed from growth media and washed twice with isotonic saline consisting of 85 mm NaCl, 5 mm KCl, 1.5 mm CaCl 2, 1 mm MgCl 2, 5 mm Glucose, 90 mm mannitol, HEPES (ph7.5). Cells were incubated with toc-fapt (0.21 µm) in isotonic saline for 30 min at 4 C. Cells were stimulated with thrombin-containing hypotonic saline consisting of 85 mm NaCl, 5 mm KCl, 1.5 mm CaCl 2, 1 mm MgCl 2, 5 mm Glucose, 5U/ml S10

11 thrombin, HEPES (ph7.5). Fluorescence images were obtained as in the imaging of Ade release from rat astrocytes. S11

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