Quantitative imaging of glutathione in live cells using a reversible reaction-based ratiometric fluorescent probe.

Materials All the chemicals were purchased from Sigma-Aldrich and Alfa Aesar unless otherwise specified. All solvents and reagents were used as obtained without further purification. Polystyrene beads (4.5 μm, catalog # 17135–5) were purchased from Polysciences Inc. All the organelle specific dyes were purchased from Thermo Fisher Scientific Inc.

Instrumentation

NMR spectra were recorded on a Varian NMR ( 1 H at 400 MHz) spectrometer. Chemical shifts (δ) were given in parts per million with reference to solvent signals [ 1 H NMR: CDCl 3 (7.26), CD 3 OD (3.31)]. UV–vis measurements were performed with a 2 × 10 mm quartz cuvette in a Cary 60 UV–vis spectrometer. Fluorescence measurements were performed with the same quartz cuvette in a Cary Eclipse fluorescence spectrophotometer with an excitation slit of 5 nm, an emission slit of 10 nm, and PMT at 650 V. Flash chromatography was performed on a Teledyne ISCO CombiFlashRf200. HPLC measurements were performed on Agilent Infinity 1200 HPLC with inline diode array and ESI-MS detectors. An Olympus FV1000 laser scanning confocal microscope system was used for cell imaging. ESI mass spectrometry was measured on a BrukerMS microTOF ESI, at the Shared Equipment Authority at Rice University. Flow cytometry was performed on BD LSR II Flow Cytometer at BCM core facilities.

Chemical Synthesis and Characterization

Refer to the SI for details.

Determination of Equilibrium Constant of TQ

Green and GSH Reaction TQ Green was dissolved in PBS buffer (10 mM, pH 7.4) containing 1% DMSO with a concentration at 32 μM. GSH was dissolved in the same PBS buffer with a concentration from 0 to 80 mM. The above solutions were mixed at 1:1 ratio, with the exception of the solution containing 80 mM of GSH and TQ Green, which was prepared by directly mixing TQ Green stock solution in DMSO with a 80 mM of GSH solution at a ratio of 1:100. All solutions were protected with nitrogen and stored in a glovebox to prevent any oxygen entering the solution. Samples of all solutions were taken out at 1, 18, 72, and 144 h after mixing. UV–vis and fluorescence were measured for all solutions.

Cell Culture and Treatment for Imaging

All cell lines used in this study were purchased from American Type Culture Collection (ATCC) and grown in DMEM (Gibco, 11965) media supplemented with 10% FBS and 1% 1003 Pen Strep (Gibco). Cells were cultured under a controlled atmosphere (37 °C, 5% CO 2 ). Glass dishes were used for the cell culture according to confocal scanning requirements. Cells were treated with TQ Green-AM (20 nM to 1 μM with 0.0025–1% DMSO in PBS) for 30 min, followed by two washing steps with trypan blue and PBS prior to imaging. Fluorescent images were acquired with a 405 nm laser/430–470 nm emission filter and 488 nm laser/575–620 nm emission filter. All the microscope settings were kept consistent in each experiment.

Calibration for Confocal Microscopy

GSH solutions (0–80 mM in PBS (10 mM, pH 7.4)) were prepared and mixed with TQ Green solution (5 μM final concentration). The above solutions were further mixed with a suspension containing 4.5 μm polystyrene beads. Cover glasses were used to hold the solutions for confocal microscopy. The same microscope settings were adopted from prior experiments.

Subcellular Colocalization Imaging

HeLa cells were cultured on glass bottom dishes as described before. For endosome labeling, cells were transfected with pCMV:mRFP-Rab5 plasmid 24 h prior to imaging; for mitochondria labeling, cells were treated with 100 nM MitoTracker Red CMXRos (Thermo Fisher Scientific Inc., catalog# M-7512) in PBS 30 min prior to imaging; for ER labeling, cells were treated with 1 μM ER-Tracker Red CMXRos (Thermo Fisher Scientific Inc., catalog# E34250) in PBS 30 min prior to imaging; for lysosome labeling, cells were treated with 50 nM LysoTracker Red DND-99 (Thermo Fisher Scientific Inc., catalog# L-7528) in PBS 120 min prior to imaging. All cells were also costained with 1 μM TQ Green-AM in PBS 30 min prior to imaging. Fluorescent images were acquired with a 405 nm laser/430–470 nm filter, 488 nm laser/505–545 nm filter, and 559 nm laser/575–620 nm filter.

Glutathione Reductase Assay and Cell Volume Measurement

The procedure was adopted from the literature with minor modification. 48 All cells were grown on six-well plates until the cell number reached about 5 × 10 5 in each well before harvesting. Cells were washed with cold PBS buffer twice and digested by 0.25 mL of trypsin under RT (treatment time varies, usually around 5 min). Then, 1 mL of fresh medium was added to neutralize, and the solution was immediately transferred to a cold 1.5 mL Eppendorf tube. A small sample was used for cell counting every time. The sample was centrifuged at 1000 g for 5 min at 4 °C, and the supernatant was discarded. The cell pellet was then washed with PBS and centrifuged again under the same conditions. The supernatant was removed, and cell-lysis buffer (1 mL 0.1% Triton-X and 0.6% sulfosalicylic acid in EDTA added PBS buffer) was added. Cells were homogenized using a Teflon pestle at 4 °C. The suspension was centrifuged at 3000 g for 4 min at 4 °C, and the supernatant was transferred to a new cold Eppendorf tube, which is ready for assay measurement. In a 96-well microtiter plate, 20 μL of cell lysate samples were placed in each well. Freshly made solutions of 5,5′-dithio-bis(2-nitrobenzoic acid) (DTNB) and glutathione reductase (GR) with concentrations of 0.33 g/L and 1.67 units/mL, respectively, were added to the same well to make the final volume 140 μL. After about 30 s, 60 μL of β-NADPH (0.67 g/L) was added, and the mixture was immediately measured for absorbance at 412 nm every 30 s for 2 min. The slope of the absorbance changes was proportional to the GSH concentration. A standard curve with known GSH concentrations was used to calibrate all the results. Cell volumes were measured by centrifuging the cell suspension (∼4 × 10 5 cells per sample) at 2500 g for 1 min in packed cell volume (PCV) tubes (Sigma-Aldrich, cat. no. Z760986).

Measurement of GSH Concentration Changes in PANC-1 Cells

PANC-1 cells were incubated with DEM (50 μM) for 2 h for stimulation of cysteine uptake. After replacement with fresh medium, cells were cultured under normal conditions for another 22 h. A separate dish of PANC-1 cells was incubated with DEM (50 μM) as an inhibitor for 24 h. TQ Green-AM (1 μM with 1% DMSO) was used to stain the cells for 30 min, followed by two washing steps with trypan blue and PBS prior to measurement. The same imaging procedures as above were performed. GSH concentrations were calculated based on the calibration curve. FACS was performed from harvested cells after the same treatment as mentioned above; fluorescent intensities were recorded with 405 nm laser/420–460 nm filter (pacific blue channel) and 488 nm laser/515–545 nm filter (FITC channel). Data were processed with FlowJo.

Materials All the chemicals were purchased from Sigma-Aldrich and Alfa Aesar unless otherwise specified. All solvents and reagents were used as obtained without further purification. Polystyrene beads (4.5 μm, catalog # 17135–5) were purchased from Polysciences Inc. All the organelle specific dyes were purchased from Thermo Fisher Scientific Inc.

Supplementary Material cb500986w_si_001.pdf