Nature Protocols: doi: /nprot Supplementary Figure 1. Fluorescent titration of probe CPDSA.
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- Annice Mercy Skinner
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1 Supplementary Figure 1 Fluorescent titration of probe CPDSA. Fluorescent titration of probe CPDSA (10 um) upon addition of GSH in HEPES (10 mm, ph = 7.4) containing 10% DMSO. Each spectrum was recorded at 20 min after the addition of GSH. (λex = 730 nm, λem = 736 nm, slit: 10/10 nm)
2 Supplementary Figure 2 Confocal microscope images following addition of probe CPDSA to HeLa cells. Confocal microscope images following addition of probe CPDSA to HeLa cells (The data is collected from FV1200, Olympus, Japan). Cell images were obtained using an excitation wavelength of 635 nm and a band path ( nm) emission filter. (A) Fluorescence image of HeLa cells; (B) Fluorescence image of HeLa cells incubated with probe CPDSA (20 μm) for 20 min; (C) Fluorescence image of HeLa cells pretreated with N-methylmaleimide (NMM, 1 mm) for 20 min and incubated with probe CPDSA (20 μm) for 20 min; (D) Fluorescence image of HeLa cells pretreated with NMM (1 mm) for 20 min, and then added with cysteine (100 μm) and incubated with probe CPDSA (20 μm) for 20 min, (E) Fluorescence image of HeLa cells pretreated with NMM (1 mm) for 20 min, and then added with homocysteine (100 μm) and incubated with probe CPDSA (20 μm) for 20 min; (F) Fluorescence image of HeLa cells pretreated with NMM (1 mm) for 20 min, and then added with GSH-MEE (100 μm) and incubated with probe CPDSA (20 μm) for 20 min. Scale bar: 15 μm.
3 Supplementary Figure 3 The time-dependent confocal microscope images following addition of probe CPDSA to HeLa cells. Confocal microscope images following addition of probe CPDSA to HeLa cells (The data is collected from FV1200, Olympus, Japan). HeLa cells were incubated with 20 μm probe CPDSA for 20 min and washed with DPBS and exchanged with new media. The time-dependent fluorescence images of probe CPDSA were acquired by confocal microscopy. Cell images were obtained using an excitation wavelength of 635 nm and a band-path ( nm) emission filter.
4 Supplementary Figure 4 The time-dependent fluorescence intensity of probe CPDSA in HeLa cells.
5 Supplementary Table 1: The comparison of fluorescent probe and bioimaging technique with other main methods for the detection of GSH. Methods Advantages Disadvantages Application Spectrophotometry High Performance Liquid Chromatography (HPLC) / Gas Chromatography (GC) High Performance Capillary Electrophoresis (HPCE) Enzymatic Cycling Electrochemistry Method Flow Cytometry Nuclear Magnetic Resonance (NMR) Fluorescence Method Our Protocol (Compared with other methods) Our Protocol (Compared with other fluorescent probes for GSH) Simple operation, high sensitivity, rapid analysis High selectivity, good stability, high accuracy Easy operation, high reliability, low detection limit High sensitivity, easy operation, determination of total glutathione Simple equipment, inexpensive, high sensitivity High sensitivity, quickly obtaining an amount of data, application in statistics Poor selectivity, high concentration More steps, longer time, low sensitivity Low sensitivity, complicated sample pretreatment, large amount of samples Poor selectivity, high requirements for sample preparation, expensive fee Poor selectivity, weak antiinterference performance, poor reproducibility Poor selectivity, defaulting suitable system detection in vitro detection in vitro and in vivo detection in vitro detection in vitro High accuracy Expensive fee Clinical diagnosis Simple operation, high sensitivity, fast detection, suited and in vivo High selectivity, high sensitivity, low detection limit, fast detection, visualization near-infrared emission, simple chemical synthesis Poor selectivity, weak antiinterference performance, background fluorescence, Chemical synthesis Photostability, keep in low temperature and in vivo and in vivo and in vivo
6 Supplementary Method: Measurement of GSH concentration by enzymatic reaction MATERIALS OxiSelect Total Glutathione (GSSG/GSH) Assay Kit (Cell BioLabs Inc. cat. no. STA 312) EQUIPMENT Spectramax Microplate Reader (Molecular devices) 96 well plate (corning, cat. no. 3591) 50 μl to 300 μl adjustable multichannel micropipette with disposable tips Conical tubes and bottles for sample and buffer preparation Sonicator Microplate reader capable of reading 405 nm REAGENT SETUP Total GSH concentration of cells was read with Microplate Reader. Absorbance at 405 nm was setted and read using the 96 well plate. Reagent setup 1X Assay Buffer: Prepare 1X Assay Buffer by adding 20 ml of deionized water to 5 ml of the 5X Assay Buffer. Mix thoroughly until homogeneous. Use this buffer for preparing kit reagents. Store at 4ºC when not in use. 1X Glutathione Reductase: Prepare the 1X Glutathione Reductase by diluting the stock solution 1:50 with 1X Assay Buffer. Vortex the stock tube thoroughly prior to preparing. Prepare only enough for immediate applications (e.g. Add 5 μl of Glutathione Reductase stock to 245 μl Assay Buffer). 1X Chromogen: Prepare the 1X Chromogen just before use. Prepare only enough for immediate applications. Dilute the Chromogen stock 1:15 with 1X Assay Diluent (e.g. Add 50 μl of Chromogen stock to 0.7 ml of 1X Assay Buffer. Vortex thoroughly. 1X NADPH: Prepare 1X NADPH by diluting the stock solution 1:50 with 1X Assay Buffer. Vortex the stock tube thoroughly prior to preparing. Prepare only enough for immediate
7 applications (e.g. Add 5 μl of NADPH stock to 245 μl Assay Buffer). Metaphosphoric Acid (MPA): Prepare a 5% (w/v) Metaphosphoric Acid (MPA) solution in deionized water. Prepare just before use. Prepare only enough for immediate applications (eg. Add 0.5 g of Metaphosphoric Acid crystals to 10 ml of deionized water). Vortex thoroughly. Note: MPA is corrosive and may cause burns. Use caution when handling acidic reagents. Procedure Determination of total GSH using the enzymatic method. Timing : 54 h We followed the manufacturer s instruction manual. same with procedure 1-8 Preparation of sample Detach adherent cells by trypsinization. Count cells and centrifuge at 500 rpm for 5 minutes at 4ºC. Wash cells with cold 1X PBS. Centrifuge suspension cells at 500 rpm for 5 minutes at 4ºC. Remove supernatant and wash cells with cold 1X PBS. Repeat centrifugation and remove solution. Immediately resuspend the pellet with μl ice-cold 5% MPA for a cell concentration of 1-5 x 10 6 cells. Mix thoroughly and homogenize or sonicate cell suspension (15sec sonicate, 30 sec in ice, 5times) and store on ice until use. Transfer the suspension to a microfuge tube and centrifuge at 12,000 rpm for 5 minutes at 4ºC and collect the supernatant. Preparation of standard curve Prepare a solution of 0.5% MPA in 1X Assay Buffer and mix. Use this solution for preparing the glutathione standards. To prepare the glutathione standards, first perform a 1:1000 dilution of the stock Glutathione Disulfide (GSSG) in 0.5% MPA/Assay Buffer. Use only enough for immediate applications (eg. Add 2 μl of Glutathione Disulfide to 1998 μl
8 0.5% MPA /Assay Buffer). This solution has a concentration of 1 μm. Use microfuge tubes to prepare a series of standards according to Table 1 below. Prepare standards fresh for each assay performed. Do not store or reuse standard preparations. Standard Tubes 1 μmgssg Standard 0.5% MPA in Assay GSSG (μm) (μl) Buffer (μl) of Tube # of Tube # of Tube # of Tube # of Tube # of Tube # Total Glutathione (GSSG/GSH) Assay Protocol Prepare and mix all reagents thoroughly before use. Prepare the glutathione standards simultaneously with the samples so they may be assayed together. Each sample, including unknown and standard, should be assayed in duplicate or triplicate. In a 96-well plate, add 25 μl of the 1X Glutathione Reductase solution to each well to be tested. Add 25 μl of the 1X NADPH solution to each well to be tested. Add 100 μl of the prepared glutathione standards or samples to each well to be tested. Mix thoroughly. Ensure that the plate reader is prepared for a kinetic assay and is set to read at 405 nm Add 50 μl of the 1X Chromogen and mix briefly. After 10 min incubation, read absorbance at 405 nm. Calculate the concentration of standards and samples.
9 Measurement of GSH concentration by enzymatic reaction
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